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1.  A new tumour suppression mechanism by p27Kip1: EGFR down-regulation mediated by JNK/c-Jun pathway inhibition 
Biochemical Journal  2014;463(Pt 3):383-392.
p27Kip1 is a potent inhibitor of cyclin-dependent kinases that drive G1-to-S cell-cycle transition. Reduced p27Kip1 expression is prevalent in a wide range of human tumours; however, the exact mechanism(s) of p27Kip1-mediated tumour suppression remains obscure. In the present study, we identified a close inverse relationship between p27Kip1 and EGFR (epidermal growth factor receptor) expression: the parental T24 human bladder cancer cells had high p27Kip1 expression but low EGFR expression and, in striking contrast, the metastatic derivative of T24 (T24T) had low p27Kip1 expression but high EGFR expression. This relationship was also found in various human cancer tissues, and was not only just correlative but also causal; depletion of p27Kip1 in MEF (mouse embryonic fibroblast) cells resulted in markedly elevated EGFR expression, a result reproducible with an Egfr promoter-luciferase reporter in both T24 and MEF cells, suggesting transcriptional repression of EGFR by p27Kip1. Indeed, p27Kip1 was found to regulate EGFR expression via the JNK (c-Jun N-terminal kinase)/c-Jun transcription factor: p27Kip1 deficiency activated JNK/c-Jun, whereas inhibition of JNK/c-Jun by dominant-negative mutants dramatically repressed Egfr transcription. Furthermore, the proximal promoter of the Egfr gene was crucial for its transcription, where the recruiting activity of c-Jun was much greater in p27Kip1−/− cells than in p27Kip1+/+ cells. Introduction of GFP–p27Kip1 into T24T cells suppressed JNK/c-Jun activation, EGFR expression and anchorage-independent growth. The results of the present study demonstrate that p27Kip1 suppresses JNK/c-Jun activation and EGFR expression in MEFs and human bladder cancer cells, and the results obtained are consistent with those from human cancer specimens. The present study provides new insights into p27Kip1 suppression of cancer cell growth, migration and metastasis.
An inverse relationship between p27Kip1 and EGFR expression in parental T24 human bladder cancer cells and various human cancer tissues was found. Depletion of p27Kip1 in cells markedly elevated EGFR expression through transcriptional repression of Egfr by p27Kip1 via the JNK/c-Jun cascade.
doi:10.1042/BJ20140103
PMCID: PMC4209780  PMID: 25121353
bladder cancer; c-Jun N-terminal kinase (JNK)/c-Jun pathway; epidermal growth factor receptor (EGFR); p27Kip1; signal transduction pathway; AP-1, activator protein 1; BME, basal medium Eagle; CDK, cyclin-dependent kinase; DMEM, Dulbecco’s modified Eagle’s medium; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSF-1, heat-shock factor 1; Hsp, heat-shock protein; IHC, immunohistochemistry; JNK, c-Jun N-terminal kinase; MEF, mouse embryonic fibroblast; RT, reverse transcription; SP1, specificity protein 1
2.  Patterns of p57Kip2 Expression in Embryonic Rat Brain Suggest Roles in Progenitor Cell Cycle Exit and Neuronal Differentiation 
Developmental neurobiology  2009;69(1):1-21.
In developing central nervous system, a variety of mechanisms couple cell cycle exit to differentiation during neurogenesis. The cyclin-dependent kinase (CDK) inhibitor p57Kip2 controls the transition from proliferation to differentiation in many tissues, but roles in developing brain remain uncertain. To characterize possible functions, we defined p57Kip2 protein expression in embryonic day (E) 12.5 to 20.5 rat brains using immunohistochemistry combined with markers of proliferation and differentiation. p57Kip2 was localized primarily in cell nuclei and positive cells formed two distinct patterns including wide dispersion and laminar aggregation that were brain region-specific. From E12.5 to E16.5, p57Kip2 expression was detected mainly in ventricular (VZ) and/or mantle zones of hippocampus, septum, basal ganglia, thalamus, hypothalamus, midbrain and spinal cord. After E18.5, p57Kip2 was detected in select regions undergoing differentiation. p57Kip2 expression was also compared to regional transcription factors, including Ngn2, Nkx2.1 and Pax6. Time course studies performed in diencephalon showed that p57Kip2 immunoreactivity co-localized with BrdU at 8 hr in nuclei exhibiting the wide dispersion pattern, whereas co-localization in the laminar pattern occurred only later. Moreover, p57Kip2 frequently co-localized with neuronal marker, β-III tubulin. Finally, we characterized relationships of p57Kip2 to CDK inhibitor p27Kip1: In proliferative regions, p57Kip2 expression preceded p27Kip1 as cells underwent differentiation, though the proteins co-localized in substantial numbers of cells, suggesting potentially related yet distinct functions of Cip/Kip family members during neurogenesis. Our observations that p57Kip2 exhibits nuclear expression as precursors exit the cell cycle and begin expressing neuronal characteristics suggests that the CDK inhibitor contributes to regulating the transition from proliferation to differentiation during brain development.
doi:10.1002/dneu.20680
PMCID: PMC2967216  PMID: 18814313
Cyclin-Dependent Kinase Inhibitor p57Kip2; Embryonic Development/physiology; Nervous System/cytology/*embryology; Brain/embryology; Neuronal Differentiation
3.  A Novel Mutation in the Upstream Open Reading Frame of the CDKN1B Gene Causes a MEN4 Phenotype 
PLoS Genetics  2013;9(3):e1003350.
The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27KIP1, an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27KIP1 expression and/or localization are often observed in tumor cells, further confirming its central role in regulating the cell cycle. Recently, germline mutations in CDKN1B have been associated with the inherited multiple endocrine neoplasia syndrome type 4, an autosomal dominant syndrome characterized by varying combinations of tumors affecting at least two endocrine organs. In this study we identified a 4-bp deletion in a highly conserved regulatory upstream ORF (uORF) in the 5′UTR of the CDKN1B gene in a patient with a pituitary adenoma and a well-differentiated pancreatic neoplasm. This deletion causes the shift of the uORF termination codon with the consequent lengthening of the uORF–encoded peptide and the drastic shortening of the intercistronic space. Our data on the immunohistochemical analysis of the patient's pancreatic lesion, functional studies based on dual-luciferase assays, site-directed mutagenesis, and on polysome profiling show a negative influence of this deletion on the translation reinitiation at the CDKN1B starting site, with a consequent reduction in p27KIP1 expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27KIP1 activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27KIP1 activity can also be modulated by an uORF and mutations affecting uORF could change p27KIP1 expression. This study adds the CDKN1B gene to the short list of genes for which mutations that either create, delete, or severely modify their regulatory uORFs have been associated with human diseases.
Author Summary
Gene expression can be modulated at different steps on the way from DNA to protein including control of transcription, translation, and post-translational modifications. An abnormality in the regulation of mRNA and protein expression is a hallmark of many human diseases, including cancer. In some eukaryotic genes translation can be influenced by small DNA sequences termed upstream open reading frames (uORFs). These elements located upstream to the gene start codon may either negatively influence the ability of the translational machinery to reinitiate translation of the main protein or, much less frequently, stimulate protein translation by enabling the ribosomes to bypass cis-acting inhibitory elements. CDKN1B, which encodes the cell cycle inhibitor p27KIP1, includes an uORF in its 5′UTR sequence. p27KIP1 expression is often reduced in cancer, and germline mutations have been identified in CDKN1B in patients affected with a syndrome (MEN4) characterized by varying combinations of tumors in endocrine glands. Here we show that a small deletion in the uORF upstream to CDKN1B reduces translation reinitiation efficiency, leading to underexpression of p27KIP1 and coinciding with tumorigenesis. This study describes a novel mechanism by which p27KIP1 could be underexpressed in human tumors. In addition, our data provide a new insight to the unique pathogenic potential of uORFs in human diseases.
doi:10.1371/journal.pgen.1003350
PMCID: PMC3605397  PMID: 23555276
4.  Phosphorylation of p27Kip1 by JAK2 directly links cytokine receptor signaling to cell cycle control 
Oncogene  2011;30(32):3502-3512.
Janus kinase 2 (JAK2) couples ligand activation of cell surface cytokine receptors to the regulation of cellular functions including cell cycle progression, differentiation and apoptosis. It thereby coordinates biological programs such as development and hematopoiesis. Unscheduled activation of JAK2 by point mutations or chromosomal translocations can induce hyperproliferation and hematological malignancies. Typical signal transduction by the JAK2 tyrosine kinase comprises phosphorylation of STAT transcription factors. In this study, we describe the identification of the cyclin-dependent kinase (CDK) inhibitor p27Kip1 as a novel JAK2 substrate. JAK2 can directly bind and phosphorylate p27Kip1. Both, the JAK2 FERM domain and its kinase domain bind to p27Kip1. JAK2 phosphorylates tyrosine residue 88 (Y88) of p27Kip1. We previously reported that Y88 phosphorylation of p27Kip1 by oncogenic tyrosine kinases impairs p27Kip1-mediated CDK inhibition, and initiates its ubiquitin-dependent proteasomal degradation. Consistently, we now find that active oncogenic JAK2V617F reduces p27Kip1 stability and protein levels in patient-derived cell lines harboring the mutant JAK2V617F allele. Moreover, tyrosine phosphorylation of p27Kip1 is impaired and p27Kip1 expression is restored upon JAK2V617F inactivation by small hairpin RNA-mediated knockdown or by the pyridone-containing tetracycle JAK inhibitor-I, indicating that direct phosphorylation of p27Kip1 can contribute to hyperproliferation of JAK2V617F-transformed cells. Activation of endogenous JAK2 by interleukin-3 (IL-3) induces Y88 phosphorylation of p27Kip1, thus unveiling a novel link between cytokine signaling and cell cycle control in non-transformed cells. Oncogenic tyrosine kinases could use this novel pathway to promote hyperproliferation in tumor cells.
doi:10.1038/onc.2011.68
PMCID: PMC3160490  PMID: 21423214
cell cycle control; CDK inhibitors; p27Kip1; tyrosine kinases; JAK2; JAK2V617F
5.  Partial hepatectomy in rats results in immediate down-regulation of p27Kip1 in residual liver tissue by transcriptional and post-translational processes 
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.
doi:10.3389/fphys.2013.00139
PMCID: PMC3680744  PMID: 23781207
cell cycle regulator; cyclin-dependent kinase inhibitor; Kip1; compensatory growth; liver regeneration; rat hepatocytes; cell proliferation
6.  Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27kip1 
Respiratory Research  2007;8(1):77.
Background
Hypoxia induces the proliferation of pulmonary arterial smooth muscle cell (PASMC) in vivo and in vitro, and prostacyclin analogues are thought to inhibit the growth of PASMC. Previous studies suggest that p27kip1, a kind of cyclin-dependent kinase inhibitor, play an important role in the smooth muscle cell proliferation. However, the mechanism of hypoxia and the subcellular interactions between p27kip1 and prostacyclin analogues in human pulmonary arterial smooth muscle cell (HPASMC) are not fully understood.
Methods
We investigated the role of p27kip1 in the ability of Beraprost sodium (BPS; a stable prostacyclin analogue) to inhibit the proliferation of HPASMC during hypoxia. To clarify the biological effects of hypoxic air exposure and BPS on HPASMC, the cells were cultured in a hypoxic chamber under various oxygen concentrations (0.1–21%). Thereafter, DNA synthesis was measured as bromodeoxyuridine (BrdU) incorporation, the cell cycle was analyzed by flow cytometry with propidium iodide staining. The p27kip1 mRNA and protein expression and it's stability was measured by real-time RT-PCR and Western blotting. Further, we assessed the role of p27kip1 in HPASMC proliferation using p27kip1 gene knockdown using small interfering RNA (siRNA) transfection.
Results
Although severe hypoxia (0.1% oxygen) suppressed the proliferation of serum-stimulated HPASMC, moderate hypoxia (2% oxygen) enhanced proliferation in accordance with enhanced p27kip1 protein degradation, whereas BPS suppressed HPASMC proliferation under both hypoxic and normoxic conditions by suppressing p27kip1 degradation with intracellular cAMP-elevation. The 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), a cAMP analogue, had similar action as BPS in the regulation of p27kip1. Moderate hypoxia did not affect the stability of p27kip1 protein expression, but PDGF, known as major hypoxia-induced growth factors, significantly decreased p27kip1 protein stability. We also demonstrated that BPS and 8-Br-cAMP suppressed HPASMC proliferation under both hypoxic and normoxic conditions by blocking p27kip1 mRNA degradation. Furthermore, p27kip1 gene silencing partially attenuated the effects of BPS and partially restored hypoxia-induced proliferation.
Conclusion
Our study suggests that moderate hypoxia induces HPASMC proliferation, which is partially dependent of p27kip1 down-regulation probably via the induction of growth factors such as PDGF, and BPS inhibits both the cell proliferation and p27kip1 mRNA degradation through cAMP pathway.
doi:10.1186/1465-9921-8-77
PMCID: PMC2164950  PMID: 17974037
7.  Streptogramin- and tetracycline-responsive dual regulated expression of p27Kip1 sense and antisense enables positive and negative growth control of Chinese hamster ovary cells 
Nucleic Acids Research  2001;29(4):e19.
We constructed a dual regulated expression vector cassette (pDuoRex) whereby two heterologous genes can be independently regulated via streptogramin- and tetracycline-responsive promoters. Two different constructs containing growth-promoting and growth-inhibiting genes were stably transfected in recombinant Chinese hamster ovary (CHO) cells that express the streptogramin- and tetracycline-dependent transactivators in a dicistronic configuration. An optimally balanced heterologous growth control scenario was achieved by reciprocal expression of the growth-inhibiting human cyclin-dependent kinase inhibitor p27Kip1 in sense (p27Kip1S) and antisense (p27Kip1AS) orientation. Exclusive expression of p27Kip1S resulted in complete G1-phase-specific growth arrest, while expression of only p27Kip1AS showed significantly increased proliferation compared to control cultures (both antibiotics present), presumably by decreasing host cell p27Kip1 expression. In a second system, a derivative of pDuoRex encoding streptogramin-responsive expression of the growth-promoting SV40 small T antigen (sT) and tetracycline-regulated expression of p27Kip1 was stably transfected into CHO cells. Expression of sT alone resulted in an increase in cell proliferation, but the expression of p27Kip1 failed to provide the expected G1-specific growth arrest despite having demonstrated expression of the protein. This illustrates the difficulty in balancing the complex pathways underlying cell proliferation control through the expression of two functionally distinct genes involved in those pathways, and how a single-gene sense/antisense approach using pDuoRex can overcome this barrier to complete metabolic engineering control.
PMCID: PMC29626  PMID: 11160939
8.  Cortactin Modulates RhoA Activation and Expression of Cip/Kip Cyclin-Dependent Kinase Inhibitors To Promote Cell Cycle Progression in 11q13-Amplified Head and Neck Squamous Cell Carcinoma Cells ▿ †  
Molecular and Cellular Biology  2010;30(21):5057-5070.
The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21WAF1/Cip1, p27Kip1, and p57Kip2 and inhibition of S-phase entry. These effects were associated with increased binding of p21WAF1/Cip1 and p27Kip1 to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21WAF1/Cip1 and p27Kip1 at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21WAF1/Cip1, p27Kip1, and p57Kip2 downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27Kip1 and p57Kip2 for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.
doi:10.1128/MCB.00249-10
PMCID: PMC2953065  PMID: 20805359
9.  The Cyclin-Dependent Kinase Inhibitor p57Kip2 Regulates Cell Cycle Exit, Differentiation, and Migration of Embryonic Cerebral Cortical Precursors 
Cerebral Cortex (New York, NY)  2011;21(8):1840-1856.
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.
doi:10.1093/cercor/bhq254
PMCID: PMC3138513  PMID: 21245411
gliogenesis; in utero electroporation; neurite outgrowth; neurogenesis; transfection
10.  Exdpf Is a Key Regulator of Exocrine Pancreas Development Controlled by Retinoic Acid and ptf1a in Zebrafish 
PLoS Biology  2008;6(11):e293.
Both endocrine and exocrine pancreatic cells arise from pancreatic-duodenal homeobox 1 (pdx1)-positive progenitors. The molecular mechanisms controlling cell fate determination and subsequent proliferation, however, are poorly understood. Unlike endocrine cells, less is known about exocrine cell specification. We report here the identification and characterization of a novel exocrine cell determinant gene, exocrine differentiation and proliferation factor (exdpf), which is highly expressed in the exocrine cell progenitors and differentiated cells of the developing pancreas in zebrafish. Knockdown of exdpf by antisense morpholino caused loss or significant reduction of exocrine cells due to lineage-specific cell cycle arrest but not apoptosis, whereas the endocrine cell mass appeared normal. Real-time PCR results demonstrated that the cell cycle arrest is mediated by up-regulation of cell cycle inhibitor genes p21Cip, p27Kip, and cyclin G1 in the exdpf morphants. Conversely, overexpression of exdpf resulted in an overgrowth of the exocrine pancreas and a severe reduction of the endocrine cell mass, suggesting an inhibitory role for exdpf in endocrine cell progenitors. We show that exdpf is a direct target gene of pancreas-specific transcription factor 1a (Ptf1a), a transcription factor critical for exocrine formation. Three consensus Ptf1a binding sites have been identified in the exdpf promoter region. Luciferase assay demonstrated that Ptf1a promotes transcription of the exdpf promoter. Furthermore, exdpf expression in the exocrine pancreas was lost in ptf1a morphants, and overexpression of exdpf successfully rescued exocrine formation in ptf1a-deficient embryos. Genetic evidence places expdf downstream of retinoic acid (RA), an instructive signal for pancreas development. Knocking down exdpf by morpholino abolished ectopic carboxypeptidase A (cpa) expression induced by RA. On the other hand, exdpf mRNA injection rescued endogenous cpa expression in embryos treated with diethylaminobenzaldehyde, an inhibitor of RA signaling. Moreover, exogenous RA treatment induced anterior ectopic expression of exdpf and trypsin in a similar pattern. Our study provides a new understanding of the molecular mechanisms controlling exocrine cell specification and proliferation by a novel gene, exdpf. Highly conserved in mammals, the expression level of exdpf appears elevated in several human tumors, suggesting a possible role in tumor pathogenesis.
Author Summary
The pancreas is a vital organ comprising endocrine and exocrine components. Both endocrine and exocrine cells derive from a common pool of progenitors present in the gut endoderm during embryogenesis. The molecular mechanisms regulating cell fate decisions and lineage-specific proliferation are not fully understood. In this work, we report the characterization of a novel gene, exocrine differentiation and proliferation factor (exdpf), as a regulator for exocrine cell fate and differentiation/proliferation. We show that it is a direct target of the transcription factor pancreas-specific transcription factor 1a (Ptf1a), which is expressed in progenitors that give rise to all pancreatic cell types. We find that a deficiency of exdpf results in a severe reduction of exocrine size due to defects in cell proliferation. Consistent with this finding, overexpression of exdpf leads to an increase of exocrine size and a decrease of endocrine size, suggesting a possible change in fate of the endocrine progenitors. The human ortholog of exdpf is highly conserved and its expression level appears elevated in several cancers, including hepatic and pancreatic cancers, implying a possible role in pathogenesis of these malignancies.
The zebrafishexdpf, a novel regulator of pancreatic exocrine cell fate, is essential for exocrine cell differentiation and proliferation.
doi:10.1371/journal.pbio.0060293
PMCID: PMC2586380  PMID: 19067490
11.  Homeodomain Transcription Factor Phox2a, via Cyclic AMP-Mediated Activation, Induces p27Kip1 Transcription, Coordinating Neural Progenitor Cell Cycle Exit and Differentiation▿ †  
Molecular and Cellular Biology  2006;26(23):8826-8839.
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.
doi:10.1128/MCB.00575-06
PMCID: PMC1636809  PMID: 16982676
12.  A mechanistic role for the chromatin modulator, NAP1L1, in pancreatic neuroendocrine neoplasm proliferation and metastases 
Background
The chromatin remodeler NAP1L1, which is upregulated in small intestinal neuroendocrine neoplasms (NENs), has been implicated in cell cycle progression. As p57Kip2 (CDKN1C), a negative regulator of proliferation and a tumor suppressor, is controlled by members of the NAP1 family, we tested the hypothesis that NAP1L1 may have a mechanistic role in regulating pancreatic NEN proliferation through regulation of p57Kip2.
Results
NAP1L1 silencing (siRNA and shRNA/lipofectamine approach) decreased proliferation through inhibition of mechanistic (mammalian) target of rapamycin pathway proteins and their phosphorylation (p < 0.05) in the pancreatic neuroendocrine neoplasm cell line BON in vitro (p < 0.0001) and resulted in significantly smaller (p < 0.05) and lighter (p < 0.05) tumors in the orthotopic pancreatic NEN mouse model. Methylation of the p57 Kip2 promoter was decreased by NAP1L1 silencing (p < 0.05), and expression of p57Kip2 (transcript and protein) was upregulated. For methylation of the p57 Kip2 promoter, NAP1L1 bound directly to the promoter (−164 to +21, chromatin immunoprecipitation). In 43 pancreatic NEN samples (38 primaries and 5 metastasis), NAP1L1 was over-expressed in metastasis (p < 0.001), expression which was inversely correlated with p57Kip2 (p < 0.01) on mRNA and protein levels. Menin was not differentially expressed.
Conclusion
NAP1L1 is over-expressed in pancreatic neuroendocrine neoplasm metastases and epigenetically promotes cell proliferation through regulation of p57 Kip2 promoter methylation.
doi:10.1186/1756-8935-7-15
PMCID: PMC4112619  PMID: 25071868
NAP1L1; Pancreatic neuroendocrine neoplasms; pNETs; Promoter methylation; p57; Proliferation
13.  Forkhead Transcription Factor FKHR-L1 Modulates Cytokine-Dependent Transcriptional Regulation of p27KIP1 
Molecular and Cellular Biology  2000;20(24):9138-9148.
Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27KIP1 through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27KIP1 promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27KIP1 appears to be critical in the regulation of cell survival since mere ectopic expression of p27KIP1 was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27KIP1 null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27KIP1 transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.
PMCID: PMC102172  PMID: 11094066
14.  A single-nucleotide polymorphism in the human p27kip1 gene (-838C>A) affects basal promoter activity and the risk of myocardial infarction 
BMC Biology  2004;2:5.
Background
Excessive proliferation of vascular smooth muscle cells and leukocytes within the artery wall is a major event in the development of atherosclerosis. The growth suppressor p27kip1 associates with several cyclin-dependent kinase/cyclin complexes, thereby abrogating their capacity to induce progression through the cell cycle. Recent studies have implicated p27kip1 in the control of neointimal hyperplasia. For instance, p27kip1 ablation in apolipoprotein-E-null mice enhanced arterial cell proliferation and accelerated atherogenesis induced by dietary cholesterol. Therefore, p27kip1 is a candidate gene to modify the risk of developing atherosclerosis and associated ischaemic events (i.e., myocardial infarction and stroke).
Results
In this study we found three common single-nucleotide polymorphisms in the human p27kip1 gene (+326T>G [V109G], -79C>T, and -838C>A). The frequency of -838A carriers was significantly increased in myocardial infarction patients compared to healthy controls (odds ratio [OR] = 1.73, 95% confidence interval [95%CI] = 1.12–2.70). In addition, luciferase reporter constructs driven by the human p27kip1 gene promoter containing A at position -838 had decreased basal transcriptional activity when transiently transfected in Jurkat cells, compared with constructs bearing C in -838 (P = 0.04).
Conclusions
These data suggest that -838A is associated with reduced p27kip1 promoter activity and increased risk of myocardial infarction.
doi:10.1186/1741-7007-2-5
PMCID: PMC400507  PMID: 15061869
myocardial infarction; p27kip1; single-nucleotide polymorphisms
15.  Regulation of p27Kip1 by Sox2 maintains quiescence of inner pillar cells in the murine auditory sensory epithelium 
Sox2 plays critical roles in cell fate specification during development and in stem cell formation; however, its role in postmitotic cells is largely unknown. Sox2 is highly expressed in supporting cells (SCs) of the postnatal mammalian auditory sensory epithelium, which unlike non-mammalian vertebrates remains quiescent even after sensory hair cell damage. Here, we induced the ablation of Sox2, specifically in SCs at three different postnatal ages (neonatal, juvenile and adult) in mice. In neonatal mice, Sox2-null inner pillar cells (IPCs, a subtype of SCs) proliferated and generated daughter cells, while other SC subtypes remained quiescent. Furthermore, p27Kip1, a cell cycle inhibitor, was absent in Sox2-null IPCs. Similarly, upon direct deletion of p27Kip1, p27Kip1-null IPCs also proliferated but retained Sox2 expression. Interestingly, cell cycle control of IPCs by Sox2-mediated expression of p27Kip1 gradually declined with age. In addition, deletion of Sox2 or p27Kip1 did not cause a cell fate change. Finally, chromatin immunoprecipitation with Sox2 antibodies and luciferase reporter assays with the p27Kip1 promoter support that Sox2 directly activates p27Kip1 transcription in postmitotic IPCs. Hence, in contrast to the well-known activity of Sox2 in promoting proliferation and cell fate determination, our data demonstrate that Sox2 plays a novel role as a key upstream regulator of p27Kip1 to maintain the quiescent state of postmitotic IPCs. Our studies suggest that manipulating Sox2 or p27Kip1 expression is an effective approach to inducing proliferation of neonatal auditory IPCs, an initial but necessary step toward restoring hearing in mammals.
doi:10.1523/JNEUROSCI.0686-12.2012
PMCID: PMC3427024  PMID: 22855803
16.  Regulation of p27Kip1 by Sox2 maintains quiescence of inner pillar cells in the murine auditory sensory epithelium 
Sox2 plays critical roles in cell fate specification during development and in stem cell formation; however, its role in postmitotic cells is largely unknown. Sox2 is highly expressed in supporting cells (SCs) of the postnatal mammalian auditory sensory epithelium, which unlike non-mammalian vertebrates remains quiescent even after sensory hair cell damage. Here, we induced the ablation of Sox2, specifically in SCs at three different postnatal ages (neonatal, juvenile and adult) in mice. In neonatal mice, Sox2-null inner pillar cells (IPCs, a subtype of SCs) proliferated and generated daughter cells, while other SC subtypes remained quiescent. Furthermore, p27Kip1, a cell cycle inhibitor, was absent in Sox2-null IPCs. Similarly, upon direct deletion of p27Kip1, p27Kip1-null IPCs also proliferated but retained Sox2 expression. Interestingly, cell cycle control of IPCs by Sox2-mediated expression of p27Kip1 gradually declined with age. In addition, deletion of Sox2 or p27Kip1 did not cause a cell fate change. Finally, chromatin immunoprecipitation with Sox2 antibodies and luciferase reporter assays with the p27Kip1 promoter support that Sox2 directly activates p27Kip1 transcription in postmitotic IPCs. Hence, in contrast to the well-known activity of Sox2 in promoting proliferation and cell fate determination, our data demonstrate that Sox2 plays a novel role as a key upstream regulator of p27Kip1 to maintain the quiescent state of postmitotic IPCs. Our studies suggest that manipulating Sox2 or p27Kip1 expression is an effective approach to inducing proliferation of neonatal auditory IPCs, an initial but necessary step toward restoring hearing in mammals.
doi:10.1523/JNEUROSCI.0686-12.2012
PMCID: PMC3427024  PMID: 22855803
17.  The homeodomain protein Cux1 interacts with Grg4 to repress p27kip1 expression during kidney development 
Gene  2009;439(1-2):87-94.
The homeodomain protein Cux1 is highly expressed in the nephrogenic zone of the developing kidney where it functions to regulate cell proliferation. Here we show that Cux1 directly interacts with the co-repressor Grg4 (Groucho 4), a known effector of Notch signaling. Promoter reporter based luciferase assays revealed enhanced repression of p27kip1 promoter activity by Cux1 in the presence of Grg4. Chromatin immunoprecipitation (ChIP) assays demonstrated the direct interaction of Cux1 with p27kip1 in newborn kidney tissue in vivo. ChIP assays also identified interactions of Cux1, Grg4, HDAC1, and HDAC3 with p27kip1 at two separate sites in the p27kip1 promoter. DNAse1 footprinting experiments revealed that Cux1 binds to the p27kip1 promoter on the sequence containing two Sp1 sites and a CCAAT box ~500 bp from the transcriptional start site, and to an AT rich sequence ~1.5 KB from the transcriptional start site. Taken together, these results identify Grg4 as an interacting partner for Cux1 and suggest a mechanism of p27kip1 repression by Cux1 during kidney development.
doi:10.1016/j.gene.2009.03.014
PMCID: PMC2742960  PMID: 19332113
18.  The Sp1 Family of Transcription Factors Is Involved in p27Kip1-Mediated Activation of Myelin Basic Protein Gene Expression 
Molecular and Cellular Biology  2003;23(12):4035-4045.
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.
doi:10.1128/MCB.23.12.4035-4045.2003
PMCID: PMC156141  PMID: 12773549
19.  Beta-estradiol attenuates hypoxic pulmonary hypertension by stabilizing the expression of p27kip1 in rats 
Respiratory Research  2010;11(1):182.
Background
Pulmonary vascular structure remodeling (PVSR) is a hallmark of pulmonary hypertension. P27kip1, one of critical cyclin-dependent kinase inhibitors, has been shown to mediate anti-proliferation effects on various vascular cells. Beta-estradiol (β-E2) has numerous biological protective effects including attenuation of hypoxic pulmonary hypertension (HPH). In the present study, we employed β-E2 to investigate the roles of p27kip1 and its closely-related kinase (Skp-2) in the progression of PVSR and HPH.
Methods
Sprague-Dawley rats treated with or without β-E2 were challenged by intermittent chronic hypoxia exposure for 4 weeks to establish hypoxic pulmonary hypertension models, which resemble moderate severity of hypoxia-induced PH in humans. Subsequently, hemodynamic and pulmonary pathomorphology data were gathered. Additionally, pulmonary artery smooth muscle cells (PASMCs) were cultured to determine the anti-proliferation effect of β-E2 under hypoxia exposure. Western blotting or reverse transcriptional polymerase chain reaction (RT-PCR) were adopted to test p27kip1, Skp-2 and Akt-P changes in rat lung tissue and cultured PASMCs.
Results
Chronic hypoxia significantly increased right ventricular systolic pressures (RVSP), weight of right ventricle/left ventricle plus septum (RV/LV+S) ratio, medial width of pulmonary arterioles, accompanied with decreased expression of p27kip1 in rats. Whereas, β-E2 treatment repressed the elevation of RVSP, RV/LV+S, attenuated the PVSR of pulmonary arterioles induced by chronic hypoxia, and stabilized the expression of p27kip1. Study also showed that β-E2 application suppressed the proliferation of PASMCs and elevated the expression of p27kip1 under hypoxia exposure. In addition, experiments both in vivo and in vitro consistently indicated an escalation of Skp-2 and phosphorylated Akt under hypoxia condition. Besides, all these changes were alleviated in the presence of β-E2.
Conclusions
Our results suggest that β-E2 can effectively attenuate PVSR and HPH. The underlying mechanism may partially be through the increased p27kip1 by inhibiting Skp-2 through Akt signal pathway. Therefore, targeting up-regulation of p27kip1 or down-regulation of Skp-2 might provide new strategies for treatment of HPH.
doi:10.1186/1465-9921-11-182
PMCID: PMC3022723  PMID: 21182801
20.  p27Kip1 and p130 Cooperate To Regulate Hematopoietic Cell Proliferation In Vivo†  
Molecular and Cellular Biology  2006;26(16):6170-6184.
To investigate the potential functional cooperation between p27Kip1 and p130 in vivo, we generated mice deficient for both p27Kip1 and p130. In p27Kip1−/−; p130−/− mice, the cellularity of the spleens but not the thymi is significantly increased compared with that of their p27Kip1−/− counterparts, affecting the lymphoid, erythroid, and myeloid compartments. In vivo cell proliferation is significantly augmented in the B and T cells, monocytes, macrophages, and erythroid progenitors in the spleens of p27Kip1−/−; p130−/− animals. Immunoprecipitation and immunodepletion studies indicate that p130 can compensate for the absence of p27Kip1 in binding to and repressing CDK2 and is the predominant CDK-inhibitor associated with the inactive CDK2 in the p27Kip1−/− splenocytes. The finding that the p27Kip1−/−; p130−/− splenic B cells are hypersensitive to mitogenic stimulations in vitro lends support to the concept that the hyperproliferation of splenocytes is not a result of the influence of their microenvironment. In summary, our findings provide genetic and molecular evidence to show that p130 is a bona fide cyclin-dependent kinase inhibitor and cooperates with p27Kip1 to regulate hematopoietic cell proliferation in vivo.
doi:10.1128/MCB.02182-05
PMCID: PMC1592787  PMID: 16880527
21.  Transcriptional activation of the histone nuclear factor P (HiNF-P) gene by HiNF-P and its Cyclin E/CDK2 responsive co-factor p220NPAT defines a novel autoregulatory loop at the G1/S phase transition 
Gene  2007;402(1-2):94-102.
Histone nuclear factor P (HiNF-P) activates histone H4 gene transcription at the G1/S phase transition upon association with its cyclin E/CDK2 responsive co-factor p220NPAT. Here we characterize the gene regulatory pathways that control the proliferation-related expression of HiNF-P. The HiNF-P locus contains a single TATA-less 0.6 kbp promoter with multiple phylogenetically conserved transcription factor recognition motifs. Transient reporter gene assays with HiNF-P promoter deletions show that there are at least three distinct activating regions (-387/-201, -201/-100 and -100/-1) that support maximal transcription. HiNF-P gene transcription is activated by SP1 through the -100/-1 domain and repressed by E2F1 through the -201/-100 domain. The multifunctional co-regulators CBP and p300 also stimulate HiNF-P gene transcription through the -201/-1 core promoter. Importantly, the HiNF-P promoter is activated by both HiNF-P and p220NPAT. This auto-regulatory activation is further enhanced by cyclin E and CDK2, while blocked by CDK inhibition (i.e., p57KIP2 p27KIP1, p21CIP). Thus, the HiNF-P gene is a key non-histone target of p220NPAT and HiNF-P. The dependence of HiNF-P gene transcription on cyclin E/CDK2/p220NPAT signaling defines a novel feed-forward loop that may sustain HiNF-P expression in proliferating cells to support the cell cycle regulated synthesis of histone H4 proteins.
doi:10.1016/j.gene.2007.07.027
PMCID: PMC2063457  PMID: 17826007
cell cycle; E2F1; SP1; p300; CBP
22.  p27Kip1 induces an accumulation of the repressor complexes of E2F and inhibits expression of the E2F-regulated genes. 
Molecular Biology of the Cell  1997;8(9):1815-1827.
p27Kip1 is an inhibitor of the cyclin-dependent kinases and it plays an inhibitory role in the progression of cell cycle through G1 phase. To investigate the mechanism of cell cycle inhibition by p27Kip1, we constructed a cell line that inducibly expresses p27Kip1 upon addition of isopropyl-1-thio-beta-D-galactopyranoside in the culture medium. Isopropyl-1-thio-beta-D-galactopyranoside-induced expression of p27Kip1 in these cells causes a specific reduction in the expression of the E2F-regulated genes such as cyclin E, cyclin A, and dihydrofolate reductase. The reduction in the expression of these genes correlates with the p27Kip1-induced accumulation of the repressor complexes of the E2F family of factors (E2Fs). Our previous studies indicated that p21WAF1 could disrupt the interaction between cyclin/cyclin-dependent kinase 2 (cdk2) and the E2F repressor complexes E2F-p130 and E2F-p107. We show that p27Kip1, like p21WAF1, disrupts cyclin/cdk2-containing complexes of E2F-p130 leading to the accumulation of the E2F-p130 complexes, which is found in growth-arrested cells. In transient transfection assays, expression of p27Kip1 specifically inhibits transcription of a promoter containing E2F-binding sites. Mutants of p27Kip1 harboring changes in the cyclin- and cdk2-binding motifs are deficient in inhibiting transcription from the E2F sites containing reporter gene. Moreover, these mutants of p27Kip1 are also impaired in disrupting the interaction between cyclin/cdk2 and the repressor complexes of E2Fs. Taken together, these observations suggest that p27Kip1 reduces expression of the E2F-regulated genes by generating repressor complexes of E2Fs. Furthermore, the results also demonstrate that p27Kip1 inhibits expression of cyclin A and cyclin E, which are critical for progression through the G1-S phases.
Images
PMCID: PMC305739  PMID: 9307976
23.  The major transcription initiation site of the p27Kip1 gene is conserved in human and mouse and produces a long 5'-UTR 
Background
The cyclin-dependent kinase inhibitor p27Kip1 is essential for proper control of cell cycle progression. The levels of p27Kip1 are regulated by several mechanisms including transcriptional and translational controls. In order to delineate the molecular details of these regulatory mechanisms it is important to identify the transcription initiation site within the p27Kip1 gene, thereby defining the promoter region of the gene and the 5'-untranslated region of the p27Kip1 mRNA. Although several previous studies have attempted to map p27Kip1 transcription start sites, the results vary widely for both the mouse and human genes. In addition, even though the mouse and human p27Kip1 gene sequences are very highly conserved, the reported start sites are notably different.
Results
In this report, using a method that identifies capped ends of mRNA molecules together with RNase protection assays, we demonstrate that p27Kip1 transcription is initiated predominantly from a single site which is conserved in the human and mouse genes. Initiation at this site produces a 5'-untranslated region of 472 nucleotides in the human p27Kip1 mRNA and 502 nucleotides in the mouse p27Kip1 mRNA. In addition, several minor transcription start sites were identified for both the mouse and human genes.
Conclusions
These results demonstrate that the major transcription initiation sites in the mouse and human p27Kip1 genes are conserved and that the 5'-UTR of the p27Kip1 mRNA is much longer than generally believed. It will be important to consider these findings when designing experiments to identify elements that are involved in regulating the cellular levels of p27Kip1.
doi:10.1186/1471-2199-2-12
PMCID: PMC59625  PMID: 11696240
24.  Repression of p27kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells. 
Molecular and Cellular Biology  1996;16(8):4327-4336.
We have investigated the regulation of p27kip1, a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G0) to a proliferative (G1) state. The level of p27kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27kip1, as well as a transient increase in cyclin D1-associated p27kip1 that later declines concomitantly with the loss of total p27kip1. Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G0. Synthesis of p27kip1 as determined by incorporation of [35S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27kip1. Northern (RNA) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27kip1 mRNA, suggesting that the PDGF-regulated decrease in p27kip1 expression occurred through a translational mechanism.
PMCID: PMC231431  PMID: 8754833
25.  Distinct Developmental Roles of Cell Cycle Inhibitors p57Kip2 and p27Kip1 Distinguish Pituitary Progenitor Cell Cycle Exit from Cell Cycle Reentry of Differentiated Cells▿ † 
Molecular and Cellular Biology  2009;29(7):1895-1908.
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.
doi:10.1128/MCB.01885-08
PMCID: PMC2655618  PMID: 19139274

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