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1.  A single nucleotide polymorphism in the p27Kip1 gene is associated with primary patency of lower extremity vein bypass grafts 
Journal of vascular surgery  2013;57(5):1179-1185.e2.
Objective
Factors responsible for the variability in outcomes after lower extremity vein bypass grafting (LEVBG) are poorly understood. Recent evidence has suggested that a single nucleotide polymorphism (SNP) in the promoter region of the p27Kip1 gene, a cell-cycle regulator, is associated with coronary in-stent restenosis. We hypothesized an association with vein graft patency.
Methods
This was a retrospective genetic association study nested within a prospective cohort of 204 patients from three referral centers undergoing LEVBG for claudication or critical ischemia. The main outcome measure was primary vein graft patency.
Results
All patients were followed up for a minimum of 1 year with duplex graft surveillance (median follow-up, 893 days; interquartile range, 539-1315). Genomic DNA was isolated and SNP analysis for the p27Kip1-838C>A variants was performed. Allele frequencies were correlated with graft outcome using survival analysis and Cox proportional hazards modeling. The p27Kip1-838C>A allele frequencies observed were CA, 53%; CC, 30%; and AA, 17%, satisfying Hardy-Weinberg equilibrium. Race (P = .025) and history of coronary artery disease (P = .027) were different across the genotypes; all other baseline variables were similar. Primary graft patency was greater among patients with the −838AA genotype (75% AA vs 55% CA/CC at 3 years; P = .029). In a Cox proportional hazards model including age, sex, race, diabetes, critical limb ischemia, redo (vs primary) bypass, vein type, and baseline C-reactive protein level, the p27Kip1-838AA genotype was significantly associated with higher graft patency (hazard ratio for failure, 0.4; 95% confidence interval, 0.17-0.93). Genotype was also associated with early (0-1 month) changes in graft lumen diameter by ultrasound imaging.
Conclusions
These data suggest that the p27Kip1-838C>A SNP is associated with LEVBG patency and, together with previous reports, underscore a central role for p27Kip1 in the generic response to vascular injury.
doi:10.1016/j.jvs.2012.11.040
PMCID: PMC3644510  PMID: 23312942
2.  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
3.  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
4.  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
5.  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
6.  S-phase kinase-associated protein-2 (Skp2) promotes vascular smooth muscle cell proliferation and neointima formation in vivo 
Journal of Vascular Surgery  2009;50(5-4):1135-1142.
Objective
Vascular smooth muscle cell (VSMC) proliferation plays an important role in the development of postangioplasty or in-stent restenosis, venous graft failure, and atherosclerosis. Our previous work has demonstrated S-phase kinase-associated protein-2 (Skp2), an F-box subunit of SCFSkp2 ubiquitin ligase, as an important mediator and common final pathway for growth factors, extracellular matrices, and cyclic-nucleotides to regulate VSMC proliferation in vitro. However, whether alteration of Skp2 function also regulates VSMC proliferation in vivo and neointimal thickening postvascular injury remains unclear. We investigated the effect of Skp2 on VSMC proliferation and neointimal formation in vivo.
Methods and Results
Firstly, we demonstrated that Skp2-null mice developed significantly smaller neointimal areas than wild-type mice after carotid ligation. Secondly, to further identify a local rather than a systemic effect of Skp2 alteration, we demonstrated that adenovirus-mediated expression of dominant-negative Skp2 in the balloon-injured rat carotid artery significantly increased medial p27Kip1 levels, inhibited VSMC proliferation, and the subsequent neointimal thickening. Lastly, to determine if Skp2 alone is sufficient to drive VSMC proliferation and lesion development in vivo, we demonstrated that adenovirus-delivery of wild-type Skp2 to the minimally-injured rat carotids is sufficient to downregulate p27Kip1 protein levels, enhanced medial VSMC proliferation, and the neointimal thickening.
Conclusion
This data provides, we believe for the first time, a more comprehensive understanding of Skp2 in the regulation of VSMC proliferation and neointimal formation and suggests that Skp2 is a promising target in the treatment of vasculoproliferative diseases.
Clinical Relevance
This manuscript describes our latest work investigating the role of the Skp2, an F-box protein component of the SCFskp2 ubiquitin-ligase, in promoting VSMC proliferation, and neointima formation in response to vascular injury in vivo. Our previous work has identified a major role for Skp2 as a key target for numerous positive and negative growth regulatory signals in vitro. These signals converge to regulate the expression of Skp2, which then controls cell-cycle progression by promoting degradation of the cyclin-dependent kinase inhibitor, p27Kip1. Until now, there has been no data in the literature on the role played by Skp2 in the regulation of VSMC proliferation and neointima formation in vivo. Our current manuscript describes, we believe for the first time, the important role played by Skp2 in these processes, using both mouse and rat arterial injury models. This is important because proliferation of VSMCs underlies the development of postangioplasty or post-stenting restenosis, venous graft failure, and transplant arteriosclerosis. Our work demonstrates for the first time that Skp2 is a major regulator of VSMC proliferation and neointimal thickening in vivo in response to vascular injury and highlights Skp2 as a potential target for future strategies designed to combat vasculoproliferative diseases.
doi:10.1016/j.jvs.2009.07.066
PMCID: PMC2774860  PMID: 19878790
7.  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
8.  ErbB2 Potentiates Breast Tumor Proliferation through Modulation of p27Kip1-Cdk2 Complex Formation: Receptor Overexpression Does Not Determine Growth Dependency 
Molecular and Cellular Biology  2000;20(9):3210-3223.
Overexpression of the ErbB2 receptor, a major component of the ErbB receptor signaling network, contributes to the development of a number of human cancers. ErbB2 presents itself, therefore, as a target for antibody-mediated therapies. In this respect, anti-ErbB2 monoclonal antibody 4D5 specifically inhibits the growth of tumor cells overexpressing ErbB2. We have analyzed the effect of 4D5-mediated ErbB2 inhibition on the cell cycle of the breast tumor cell line BT474. 4D5 treatment of BT474 cells resulted in a G1 arrest, preceded by rapid dephosphorylation of ErbB2, inhibition of cytoplasmic signal transduction pathways, accumulation of the cyclin-dependent kinase inhibitor p27Kip1, and inactivation of cyclin-Cdk2 complexes. Time courses demonstrated that 4D5 treatment redirects p27Kip1 onto Cdk2 complexes, an event preceding increased p27Kip1 expression; this correlates with the downregulation of c-Myc and D-type cyclins (proteins involved in p27Kip1 sequestration) and the loss of p27Kip1 from Cdk4 complexes. Similar events were observed in ErbB2-overexpressing SKBR3 cells, which exhibited reduced proliferation in response to 4D5 treatment. Here, p27Kip1 redistribution resulted in partial Cdk2 inactivation, consistent with a G1 accumulation. Moreover, p27Kip1 protein levels remained constant. Antisense-mediated inhibition of p27Kip1 expression in 4D5-treated BT474 cells further demonstrated that in the absence of p27Kip1 accumulation, p27Kip1 redirection onto Cdk2 complexes is sufficient to inactivate Cdk2 and establish the G1 block. These data suggest that ErbB2 overexpression leads to potentiation of cyclin E-Cdk2 activity through regulation of p27Kip1 sequestration proteins, thus deregulating the G1/S transition. Moreover, through comparison with an ErbB2-overexpressing cell line insensitive to 4D5 treatment, we demonstrate the specificity of these cell cycle events and show that ErbB2 overexpression alone is insufficient to determine the cellular response to receptor inhibition.
PMCID: PMC85615  PMID: 10757805
9.  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
10.  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
11.  Vitamin E δ-Tocotrienol Induces p27Kip1-Dependent Cell-Cycle Arrest in Pancreatic Cancer Cells via an E2F-1-Dependent Mechanism 
PLoS ONE  2013;8(2):e52526.
Vitamin E δ-tocotrienol has been shown to have antitumor activity, but the precise molecular mechanism by which it inhibits the proliferation of cancer cells remains unclear. Here, we demonstrated that δ-tocotrienol exerted significant cell growth inhibition pancreatic ductal cancer (PDCA) cells without affecting normal human pancreatic ductal epithelial cell growth. We also showed that δ-tocotrienol-induced growth inhibition occurred concomitantly with G1 cell-cycle arrest and increased p27Kip1 nuclear accumulation. This finding is significant considering that loss of nuclear p27Kip1 expression is a well-established adverse prognostic factor in PDCA. Furthermore, δ-tocotrienol inactivated RAF-MEK-ERK signaling, a pathway known to suppress p27Kip1 expression. To determine whether p27Kip1 induction is required for δ-tocotrienol inhibition of PDCA cell proliferation, we stably silenced the CDKN1B gene, encoding p27Kip1, in MIAPaCa-2 PDCA cells and demonstrated that p27Kip1 silencing suppressed cell-cycle arrest induced by δ-tocotrienol. Furthermore, δ-tocotrienol induced p27Kip1 mRNA expression but not its protein degradation. p27Kip1 gene promoter activity was induced by δ-tocotrienol through the promoter's E2F-1 binding site, and this activity was attenuated by E2F-1 depletion using E2F-1 small interfering RNA. Finally, decreased proliferation, mediated by Ki67 and p27Kip1 expression by δ-tocotrienol, was confirmed in vivo in a nude mouse xenograft pancreatic cancer model. Our findings reveal a new mechanism, dependent on p27Kip1 induction, by which δ-tocotrienol can inhibit proliferation in PDCA cells, providing a new rationale for p27Kip1 as a biomarker for δ-tocotrienol efficacy in pancreatic cancer prevention and therapy.
doi:10.1371/journal.pone.0052526
PMCID: PMC3564846  PMID: 23393547
12.  p27KIP1 Deletions in Childhood Acute Lymphoblastic Leukemia 
Neoplasia (New York, N.Y.)  1999;1(3):253-261.
Abstract
The p27KIP1 gene, which encodes a cyclin-dependent kinase (CDK) inhibitor, has been assigned to chromosome band 12p12, a region often affected by cytogenetically apparent deletions or translocations in childhood acute lymphoblastic leukemia (ALL). As described here, fluorescence in situ hybridization (FISH) analysis of 35 primary ALL samples with cytogenetic evidence of 12p abnormalities revealed hemizygous deletions of p27KIP1 in 29 cases. Further analysis of 19 of these cases with two additional gene-specific probes from the 12p region (hematopoietic cell phosphatase, HCP and cyclin D2, CCND2) showed that p27KIP1 is located more proximally on the short arm of chromosome 12 and is deleted more frequently than either HCP or CCND2. Of 16 of these cases with hemizygous deletion of p27KIP1, only eight showed loss of HCP or CCND2, whereas loss of either of the latter two loci was uniformly associated with loss of p27KIP1. Missense mutations or mutations leading to premature termination codons were not detected in the coding sequences of the retained p27KIP1 alleles in any of the 16 ALL cases examined, indicating a lack of homozygous inactivation. By Southern blot analysis, one case of primary T-cell ALL had hemizygous loss of a single p27KIP1 allele and a 34.5-kb deletion, including the second coding exon of the other allele. Despite homozygous inactivation of p27KIP1 in this case, our data suggest that haploinsufficiency for p27KIP1 is the primary consequence of 12p chromosomal deletions in childhood ALL. The oncogenic role of reduced, but not absent, levels of p27KIP1 is supported by recent studies in murine models and evidence that this protein not only inhibits the activity of complexes containing CDK2 and cyclin E, but also promotes the assembly and catalytic activity of CDK4 or CDK6 in complexes with cyclin D.
PMCID: PMC1508076  PMID: 10935480
acute lymphoblastic leukemia; p27KIP1; cell cycle inhibitor
13.  AMPKα2 Deletion Exacerbates Neointima Formation by Upregulating Skp2 in Vascular Smooth Muscle Cells 
Circulation research  2011;109(11):1230-1239.
Rationale
Adenosine monophosphate-activated protein kinase (AMPK), a metabolic and redox sensor, is reported to suppress cell proliferation of non-malignant and tumor cells. Whether AMPKα alters vascular neointima formation induced by vascular injury is unknown.
Objective
The aim of this study was to determine the roles of AMPKα in the development of vascular neointima hyperplasia and to elucidate the underlying mechanisms.
Methods and Results
Vascular smooth muscle cells (VSMCs) proliferation and neointimal hyperplasia were evaluated in cultured VSMCs and wire-injured mouse carotid arteries from wild-type (WT, C57BL/6J), AMPKα2−/−, and AMPKα1−/− VSMCs. Mouse VSMCs derived from aortas of AMPKα2−/− mice exhibited increased proliferation compared to either WT or AMPKα1−/− VSMCs. Further, deletion of AMPKα2, but not AMPKα1, reduced the level of p27Kip1, acyclin-dependent kinase inhibitor, and increased the level of S-phase kinase-associated protein 2 (Skp2), a known E3 ubiquitin ligase for p27Kip1, via activation of p52 nuclear factor kappa B (NF-κB)-2. Moreover, either pharmacological (i.e., via compound C) or genetical (i.e., via AMPKα2-specific siRNA) inhibition of AMPK decreased p27Kip1 levels, but increased the abundance of Skp2 in human VSMCs. Furthermore, gene silencing of Skp2 reversed the levels of p27Kip1 and VSMCs proliferation. Finally, neointima formation after mechanical arterial injury was increased in AMPKα2−/−, but not AMPKα1−/−, mice.
Conclusions
These findings indicate that deletion of AMPKα2 via p52-Skp2-mediated ubiquintination and degradation of p27Kip1 accentuates neointimal hyperplasia in response to wire injury.
doi:10.1161/CIRCRESAHA.111.250423
PMCID: PMC3235405  PMID: 21980125
Neointima formation; AMPK; VSMC; NF-κB; Skp2
14.  Phosphorylation and Subcellular Localization of p27Kip1 Regulated by Hydrogen Peroxide Modulation in Cancer Cells 
PLoS ONE  2012;7(9):e44502.
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.
doi:10.1371/journal.pone.0044502
PMCID: PMC3435274  PMID: 22970236
15.  p21cip1 Degradation in Differentiated Keratinocytes Is Abrogated by Costabilization with Cyclin E Induced by Human Papillomavirus E7 
Journal of Virology  2001;75(13):6121-6134.
The human papillomavirus (HPV) E7 protein promotes S-phase reentry in a fraction of postmitotic, differentiated keratinocytes. Here we report that these cells contain an inherent mechanism that opposes E7-induced DNA replication. In organotypic raft cultures of primary human keratinocytes, neither cyclin E nor p21cip1 is detectable in situ. However, E7-transduced differentiated cells not in S phase accumulate abundant cyclin E and p21cip1. We show that normally p21cip1 protein is rapidly degraded by proteasomes. In the presence of E7 or E6/E7, p21cip1, cyclin E, and cyclin E2 proteins were all up-regulated. The accumulation of p21cip1 protein is a posttranscriptional event, and ectopic cyclin E expression was sufficient to trigger it. In constract, cdk2 and p27kip1 were abundant in normal differentiated cells and were not significantly affected by E7. Cyclin E, cdk2, and p21cip1 or p27kip1 formed complexes, and relatively little kinase activity was found associated with cyclin E or cdk2. In patient papillomas and E7 raft cultures, all p27kip1-positive cells were negative for bromodeoxyuridine (BrdU) incorporation, but only some also contained cyclin E and p21cip1. In contrast, all cyclin E-positive cells also contained p27kip1. When the expression of p21cip1 was reduced by rottlerin, a PKC δ inhibitor, p27kip1- and BrdU-positive cells remained unchanged. These observations show that high levels of endogenous p27kip1 can prevent E7-induced S-phase reentry. This inhibition then leads to the stabilization of cyclin E and p21cip1. Since efficient initiation of viral DNA replication requires cyclin E and cdk2, its inhibition accounts for heterogeneous viral activities in productively infected lesions.
doi:10.1128/JVI.75.13.6121-6134.2001
PMCID: PMC114328  PMID: 11390614
16.  Differential Regulation of P27Kip1 Expression by Mitogenic and Hypertrophic Factors 
The Journal of Cell Biology  2000;148(3):543-556.
Platelet-derived growth factor-BB (PDGF-BB) acts as a full mitogen for cultured aortic smooth muscle cells (SMC), promoting DNA synthesis and cell proliferation. In contrast, angiotensin II (Ang II) induces cellular hypertrophy as a result of increased protein synthesis, but is unable to drive cells into S phase. In an effort to understand the molecular basis for this differential growth response, we have examined the downstream effects of PDGF-BB and Ang II on regulators of the cell cycle machinery in rat aortic SMC. Both PDGF-BB and Ang II were found to stimulate the accumulation of G1 cyclins with similar kinetics. In addition, little difference was observed in the expression level of their catalytic partners, Cdk4 and Cdk2. However, while both factors increased the enzymatic activity of Cdk4, only PDGF-BB stimulated Cdk2 activity in late G1 phase. The lack of activation of Cdk2 in Ang II-treated cells was causally related to the failure of Ang II to stimulate phosphorylation of the enzyme on threonine and to downregulate p27Kip1 expression. By contrast, exposure to PDGF-BB resulted in a progressive and dramatic reduction in the level of p27Kip1 protein. The time course of p27Kip1 decline was correlated with a reduced rate of synthesis and an increased rate of degradation of the protein. Importantly, the repression of p27Kip1 synthesis by PDGF-BB was associated with a marked attenuation of Kip1 gene transcription and a corresponding decrease in Kip1 mRNA accumulation. We also show that the failure of Ang II to promote S phase entry is not related to the autocrine production of transforming growth factor-β1 by aortic SMC. These results identify p27Kip1 as an important regulator of the phenotypic response of vascular SMC to mitogenic and hypertrophic stimuli.
PMCID: PMC2174813  PMID: 10662779
growth factors; cell cycle; CDK inhibitors; gene expression; smooth muscle cells
17.  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
18.  p57KIP2 control of actin cytoskeleton dynamics is responsible for its mitochondrial pro-apoptotic effect 
Cell Death & Disease  2012;3(5):e311-.
p57 (Kip2, cyclin-dependent kinase inhibitor 1C), often found downregulated in cancer, is reported to hold tumor suppressor properties. Originally described as a cyclin-dependent kinase (cdk) inhibitor, p57KIP2 has since been shown to influence other cellular processes, beyond cell cycle regulation, including cell death and cell migration. Inhibition of cell migration by p57KIP2 is attributed to the stabilization of the actin cytoskeleton through the activation of LIM domain kinase-1 (LIMK-1). Furthermore, p57KIP2 is able to enhance mitochondrial-mediated apoptosis. Here, we report that the cell death promoting effect of p57KIP2 is linked to its effect on the actin cytoskeleton. Indeed, whereas Jasplakinolide, an actin cytoskeleton-stabilizing agent, mimicked p57KIP2's pro-apoptotic effect, destabilizing the actin cytoskeleton with cytochalsin D reversed p57KIP2's pro-apoptotic function. Conversely, LIMK-1, the enzyme mediating p57KIP2's effect on the actin cytoskeleton, was required for p57KIP2's death promoting effect. Finally, p57KIP2-mediated stabilization of the actin cytoskeleton was associated with the displacement of hexokinase-1, an inhibitor of the mitochondrial voltage-dependent anion channel, from the mitochondria, providing a possible mechanism for the promotion of the mitochondrial apoptotic cell death pathway. Altogether, our findings link together two tumor suppressor properties of p57KIP2, by showing that the promotion of cell death by p57KIP2 requires its actin cytoskeleton stabilization function.
doi:10.1038/cddis.2012.51
PMCID: PMC3366085  PMID: 22592318
p57KIP2; cell migration; cancer; cytoskeleton
19.  Expression of constitutively active 4EBP-1 enhances p27Kip1 expression and inhibits proliferation of MCF7 breast cancer cells 
Background
Eukaryotic initiation factor 4E (eIF4E) is essential for cap-dependent initiation of translation. Cell proliferation is associated with increased activity of eIF4E and elevated expression of eIF4E leads to tumorigenic transformation. Many tumors express very high levels of eIF4E and this may be a critical factor in progression of the disease. In contrast, overexpression of 4EBP, an inhibitor of eIF4E, leads to cell cycle arrest and phenotypic reversion of some transformed cells.
Results
A constitutively active form of 4EBP-1 was inducibly expressed in the human breast cancer cell line MCF7. Induction of constitutively active 4EBP-1 led to cell cycle arrest. This was not associated with a general inhibition of protein synthesis but rather with changes in specific cell cycle regulatory proteins. Cyclin D1 was downregulated while levels of the CDK inhibitor p27Kip1 were increased. The levels of cyclin E and CDK2 were unaffected but the activity of CDK2 was significantly reduced due to increased association with p27Kip1. The increase in p27Kip1 did not reflect changes in p27Kip1 mRNA or degradation rates. Rather, it was associated with enhanced synthesis of the protein, even though 4EBP-1 is expected to inhibit translation. This could be explained, at least in part, by the ability of the p27Kip1 5'-UTR to mediate cap-independent translation, which was also enhanced by expression of constitutively active 4EBP-1.
Conclusions
Expression of active 4EBP-1 in MCF7 leads to cell cycle arrest which is associated with downregulation of cyclin D1 and upregulation of p27Kip1. Upregulation of p27Kip1reflects increased synthesis which corresponds to enhanced cap-independent translation through the 5'-UTR of the p27Kip1 mRNA.
doi:10.1186/1475-2867-3-2
PMCID: PMC151675  PMID: 12633504
20.  The possible role of cell cycle regulators in multistep process of HPV-associated cervical carcinoma 
Background
Human papillomavirus (HPV) 16 and 18 are associated with cervical carcinogenesis through an interaction between HPV oncogenic proteins and cell cycle regulatory genes. However, the exact pathogenetic mechanisms are not determined yet.
Methods
We investigated 43 invasive squamous cell carcinoma (ISCC), 38 CIN III, 11 CINII and 18 CINI for cyclin D1, cyclin E, CDK4, p53, mdm-2, p21waf, p27, p16INK4A, Rb and Ki-67 aberrations using immunohistochemistry and molecular techniques. Twenty samples of normal cervical tissues (NCT) were taken as a control.
Results
There was a significant increase in the expression of Ki-67, cyclin E, CDK4, p16INK4A, Rb (p= 0.003, 0.001, 0.001, 0.01) and a significant decrease in p27KIP1 from NCT to ISCC (p = 0.003). Increased cyclin D1, p21waf, p53, mdm-2 expression, homozygous deletion (HZD) and promoter methylation (PM) of the Rb were detected in CINIII and ISCC only. On univariate analysis; tumor size, differentiation, lymph node status, FIGO stage, Ki- 67, cyclin D1, p53 and p27KIP1 are significantly associated with reduced overall survival (OS) while on multivariate analysis; only FIGO stage, Ki-67, cyclin D1, p53 and p27KIP1 were significant.
Conclusion
1) Aberrations involving p27KIP1, cyclin E, CDK4, p16INK4A are considered early events in HPV 16 and 18-associated cervical carcinoma, whereas cyclin D1 and p53 pathway abnormalities are considered late events. 2) Immunohistochemical tests for p16INK4Aand cyclin E, could help in early diagnosis of cervical carcinoma. 3) Only FIGO stage p53, cyclin D1, p27KIP1 and Ki-67 are independent prognostic factors that might help in predicting outcome of cervical cancer patients.
doi:10.1186/1472-6890-7-4
PMCID: PMC1894982  PMID: 17521451
21.  A novel role for the cyclin-dependent kinase inhibitor p27Kip1 in angiotensin II–stimulated vascular smooth muscle cell hypertrophy 
Journal of Clinical Investigation  1999;104(6):815-823.
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.
PMCID: PMC408428  PMID: 10491417
22.  E Proteins and Id2 Converge on p57Kip2 To Regulate Cell Cycle in Neural Cells 
Molecular and Cellular Biology  2006;26(11):4351-4361.
A precise balance between proliferation and differentiation must be maintained during neural development to obtain the correct proportion of differentiated cell types in the adult nervous system. The basic helix-loop-helix (bHLH) transcription factors known as E proteins and their natural inhibitors, the Id proteins, control the timing of differentiation and terminal exit from the cell cycle. Here we show that progression into S phase of human neuroblastoma cells is prevented by E proteins and promoted by Id2. Cyclin-dependent kinase inhibitors (CKI) have been identified as key effectors of cell cycle arrest in differentiating cells. However, p57Kip2 is the only CKI that is absolutely required for normal development. Through the use of global gene expression analysis in neuroblastoma cells engineered to acutely express the E protein E47 and Id2, we find that p57Kip2 is a target of E47. Consistent with the role of Id proteins, Id2 prevents activation of p57Kip2 expression, and the retinoblastoma tumor suppressor protein, a known Id2 inhibitor, counters this activity. The strong E47-mediated inhibition of entry into S phase is entirely reversed in cells in which expression of p57Kip2 is silenced by RNA interference. During brain development, expression of p57Kip2 is opposite that of Id2. Our findings identify p57Kip2 as a functionally relevant target recruited by bHLH transcription factors to induce cell cycle arrest in developing neuroblasts and suggest that deregulated expression of Id proteins may be an epigenetic mechanism to silence expression of this CKI in neural tumors.
doi:10.1128/MCB.01743-05
PMCID: PMC1489106  PMID: 16705184
23.  CDK inhibitor p57Kip2 is negatively regulated by COP9 signalosome subunit 6 
Cell Cycle  2012;11(24):4633-4641.
Subunit 6 of the COP9 signalosome complex, CSN6, is known to be critical to the regulation of the MDM2-p53 axis for cell proliferation and anti-apoptosis, but its many targets remain unclear. Here we show that p57Kip2 is a target of CSN6, and that CSN6 is a negative regulator of p57Kip2. CSN6 associates with p57Kip2, and its overexpression can decrease the steady-state expression of p57Kip2; accordingly, CSN6 deficiency leads to p57Kip2 stabilization. Mechanistic studies show that CSN6 associates with p57Kip2 and Skp2, a component of the E3 ligase, which, in turn, facilitates Skp2-mediated protein ubiquitination of p57Kip2. Loss of Skp2 compromised CSN6-mediated p57Kip2 destabilization, suggesting collaboration between Skp2 and CSN6 in degradation of p57Kip2. CSN6’s negative impact on p57Kip2 elevation translates into cell growth promotion, cell cycle deregulation and potentiated transformational activity. Significantly, univariate Kaplan-Meier analysis of tumor samples demonstrates that high CSN6 expression or low p57 expression is associated with poor overall survival. These data suggest that CSN6 is an important negative regulator of p57Kip2, and that overexpression of CSN6 in many types of cancer could lead to decreased expression of p57Kip2 and result in promoted cancer cell growth.
doi:10.4161/cc.22887
PMCID: PMC3562308  PMID: 23187808
COP9; CSN6; Skp2; cell cycle; p57
24.  Cyclin dependent kinase inhibitor p27Kip1 expression in normal and neoplastic cervical epithelium. 
Journal of Clinical Pathology  1999;52(12):880-887.
AIM: To investigate whether there is loss of the p27Kip1 protein in developing cervical cancer and whether p27Kip1 immunoreactivity has any relation to the proliferative indicator Ki-67. METHODS: The expression of p27Kip1 and Ki-67 was assessed by immunohistochemistry in serial sections from normal epithelium (13), low grade (27) and high grade (19) squamous intraepithelial lesions (LSIL, HSIL), and invasive cervical cancer (23). In the SIL cases the presence of human papillomavirus (HPV) genomic sequences was assessed by in situ hybridisation. The results were evaluated by image analysis, and reported as mean score of the percentage of p27Kip1 and of Ki-67 positive cells in each histological group. RESULTS: In general, p27Kip1 immunostaining was related to squamous differentation, and was intense in normal epithelium (47%), while it was reduced in SIL lesions as an effect of the decreased number of differentiating cells. However, decrease in the p27Kip1 expression was more evident in LSIL (36%) than in HSIL (39%); in the latter, p27Kip1 had a different intraepithelial distribution in that the staining extended to the basal cells. The average levels of p27Kip1 were similar in SIL lesions associated to low, intermediate, and high risk HPV types. Compared with normal epithelium and dysplasia, invasive cancer showed significantly lower p27Kip1 levels (23%). There was no relation between p27Kip1 and Ki-67 labelling indices in any of the histological groups examined. CONCLUSIONS: A reduction in p27Kip1 protein occurs in cervical cancer independently of the proliferative status. The changes in p27Kip1 expression may be related to the unregulated kinetics of developing cervical cancer.
Images
PMCID: PMC501653  PMID: 10711250
25.  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

Results 1-25 (1327109)