The cell cycle inhibitor p27Kip1 controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle,Brachyury and Twist

The continued turn over of human embryonic stem cells (hESC) while maintaining an undifferentiated state is dependent on the regulation of the cell cycle. Here we asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC. We identified that the protein expression of the p27^Kip1 cell cycle inhibitor is low in hESC cells and increased with differentiation. By adopting a gain and loss of function strategy we forced or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency. Using undifferentiation conditions, overexpression of p27^Kip1 in hESC lead to a G₁ phase arrest with an enlarged and flattened hESC morphology and consequent loss of self-renewal ability. Loss of p27^Kip1 caused an elongated/scatter cell-like phenotype involving upregulation of Brachyury and Twist gene expression. We demonstrate the novel finding that p27^Kip1 protein occupies the Twist1 gene promoter and manipulation of p27^Kip1 by gain and loss of function is associated with Twist gene expression changes. These results define p27^Kip1 expression levels as critical for self-renewal and pluripotency in hESC and suggest a role for p27^Kip1 in controlling an epithelial to mesenchymal transition (EMT) in hESC.     

Screening of cell cycle fusion proteins to identify kinase signaling networks

Kinase signaling networks are well-established mediators of cell cycle transitions. However, how kinases interact with the ubiquitin proteasome system (UPS) to elicit protein turnover is not fully understood. We sought a means of identifying kinase-substrate interactions to better understand signaling pathways controlling protein degradation. Our prior studies used a luciferase fusion protein to uncover kinase networks controlling protein turnover. In this study, we utilized a similar approach to identify pathways controlling the cell cycle protein p27^Kip1. We generated a p27^Kip1-luciferase fusion and expressed it in cells incubated with compounds from a library of pharmacologically active compounds. We then compared the relative effects of the compounds on p27^Kip1-luciferase fusion stabilization. This was combined with in silico kinome profiling to identify potential kinases inhibited by each compound. This approach effectively uncovered known kinases regulating p27^Kip1 turnover. Collectively, our studies suggest that this parallel screening approach is robust and can be applied to fully understand kinase-ubiquitin pathway interactions.     

CDK inhibitors,p21 and p27, participate in cell cycle exit of mammalian cardiomyocytes

Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remains largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21^Cip1 and p27^Kip1 but not p57^Kip2 showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21^Cip1 and p27^Kip1 bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21^Cip1 and p27^Kip1 knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21^Cip1 and p27^Kip play important roles in the cell cycle exit of postnatal cardiomyocytes.     

Attenuation of cell cycle regulator p27<Superscript>Kip1</Superscript> expression in vertebrate epithelial cells mediated by extracellular signals in vivo and in vitro

Cell cycle arrest in potentially dividing cells is often mediated by inhibitors of G1/S-phase cyclin-dependent kinases. The cyclin E/CDK2-inhibitor p27^Kip1 has been implicated in this context in epithelial cells. We cloned and sequenced p27^Kip1 of ducklings (Anas platyrhynchos) and used an in vitro assay system to study the mechanism of p27^Kip1 downregulation in the nasal gland which precedes an increase in proliferation rate upon initial exposure of the animals to osmotic stress. Western blot studies revealed that p27^Kip1 is downregulated during 24 h of osmotic stress in ducklings with the steepest decline in protein levels between 5 and 8 h. As indicated by the results of Northern blot and semi-quantitative PCR studies, protein downregulation is not accompanied by similar changes in mRNA levels indicating that Kip1 is regulated mainly at the translational (synthesis) or posttranslational level (degradation). Using recombinant duck Kip1 protein expressed in E. coli, we showed that Kip1 is subject to polyubiquitinylation by cytosolic enzymes from nasal gland cells indicating that loss of Kip1 may be regulated, at least in part, by acceleration of protein degradation. In cultured nasal gland tissue, attenuation of Kip1 expression could be induced by activation of the muscarinic acetylcholine receptor indicating that mAChR-receptor signalling may play a role in the re-entry of quiescent gland cells into the cell cycle.     

Connexin32 inhibits gastric carcinogenesis through cell cycle arrest and altered expression of p21Cip1 and p27Kip1

Gap junctions and their structural proteins, connexins (Cxs), have been implicated in carcinogenesis. To explore the involvement of Cx32 in gastric carcinogenesis, immunochemical analysis of Cx32 and proliferation marker Ki67 using tissue-microarrayed human gastric cancer and normal tissues was performed. In addition, after Cx32 overexpression in the human gastric cancer cell line AGS, cell proliferation, cell cycle analyses, and p21^Cip1 and p27^Kip1 expression levels were examined by bromodeoxyuridine assay, flow cytometry, real-time RT-PCR, and western blotting. Immunohistochemical study noted a strong inverse correlation between Cx32 and Ki67 expression pattern as well as their location. In vitro, overexpression of Cx32 in AGS cells inhibited cell proliferation significantly. G₁ arrest, up-regulation of cell cycle-regulatory proteins p21^Cip1 and p27^Kip1 was also found at both mRNA and protein levels. Taken together, Cx32 plays some roles in gastric cancer development by inhibiting gastric cancer cell proliferation through cell cycle arrest and cell cycle regulatory proteins. [BMB Reports 2013; 46(1): 25-30]     

p27Kip1 is required to maintain proliferative quiescence in the adult cochlea and pituitary

Cell cycle inhibitors, such as the cyclin-dependent kinase (Cdk) inhibitor proteins and retinoblastoma (Rb) family members, control exit from the cell cycle during the development of a variety of terminally differentiated tissues. It is unclear whether sustained expression of these proteins is required to prevent cell cycle re-entry in quiescent and terminally differentiated cells. The organ of Corti (cochlear sensory epithelium) and pars intermedia (intermediate lobe of the pituitary) are two tissues that share the characteristic of ongoing cell division in mice lacking either the p27^Kip1 Cdk inhibitor, Ink4 proteins or Rb. Here, we use tamoxifen-inducible mouse models to delete p27^Kip1 in postnatal animals and show this is sufficient to induce proliferation in both the organ of Corti and pars intermedia. Thus, these tissues remain sensitive to the presence of p27^Kip1 even after their developmental exit from the cell cycle. The neonatal cochlea displayed heightened sensitivity to changes in p27^Kip1 expression, with a proliferative response higher than that of constitutive null mice. In adults, the proliferative response was reduced but was accompanied by increased cell survival. In contrast, re-establishment of normal p27^Kip1 expression in animals with established pituitary tumors, in an inducible “knock-on” model, led to cessation of pituitary tumor growth, indicating the cells had maintained their susceptibility to p27-mediated growth suppression. Although restoration of p27^Kip1 did not induce apoptosis, it did lead to resolution of pathological features and normalization of gene expression. Our data underscore the importance of p27^Kip1 expression in the maintenance of cellular quiescence and terminal differentiation.Key words: proliferation, cell cycle, p27, Cdk inhibitor, auditory, cochlea, pituitary     

Prolonged mechanical ventilation induces cell cycle arrest in newborn rat lung

Rationale

The molecular mechanism(s) by which mechanical ventilation disrupts alveolar development, a hallmark of bronchopulmonary dysplasia, is unknown.

Objective

To determine the effect of 24 h of mechanical ventilation on lung cell cycle regulators, cell proliferation and alveolar formation in newborn rats.

Methods

Seven-day old rats were ventilated with room air for 8, 12 and 24 h using relatively moderate tidal volumes (8.5 mL.kg⁻¹).

Measurement and Main Results

Ventilation for 24 h (h) decreased the number of elastin-positive secondary crests and increased the mean linear intercept, indicating arrest of alveolar development. Proliferation (assessed by BrdU incorporation) was halved after 12 h of ventilation and completely arrested after 24 h. Cyclin D1 and E1 mRNA and protein levels were decreased after 8–24 h of ventilation, while that of p27^Kip1 was significantly increased. Mechanical ventilation for 24 h also increased levels of p57^Kip2, decreased that of p16^INK4a, while the levels of p21^Waf/Cip1 and p15^INK4b were unchanged. Increased p27^Kip1 expression coincided with reduced phosphorylation of p27^Kip1 at Thr¹⁵⁷, Thr¹⁸⁷ and Thr¹⁹⁸ (p<0.05), thereby promoting its nuclear localization. Similar -but more rapid- changes in cell cycle regulators were noted when 7-day rats were ventilated with high tidal volume (40 mL.kg⁻¹) and when fetal lung epithelial cells were subjected to a continuous (17% elongation) cyclic stretch.

Conclusion

This is the first demonstration that prolonged (24 h) of mechanical ventilation causes cell cycle arrest in newborn rat lungs; the arrest occurs in G₁ and is caused by increased expression and nuclear localization of Cdk inhibitor proteins (p27^Kip1, p57^Kip2) from the Kip family.     

Skp2 promotes adipocyte differentiation via a p27-independent mechanism in primary mouse embryonic fibroblasts

Skp2, the substrate-binding subunit of an SCF ubiquitin ligase complex, is a key regulator of cell cycle progression that targets substrates for degradation by the 26S proteasome. We have now shown that ablation of Skp2 in primary mouse embryonic fibroblasts (MEFs) results both in impairment of adipocyte differentiation and in the accumulation of the cyclin-dependent kinase inhibitor p27^Kip1, a principal target of the SCF^Skp2 complex. Genetic ablation of p27^Kip1 in MEFs promoted both lipid accumulation and adipocyte-specific gene expression. However, depletion of p27^Kip1 by adenovirus-mediated RNA interference failed to correct the impairment of adipocyte differentiation in Skp2^-/- MEFs. In contrast, troglitazone, a high-affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ), largely restored lipid accumulation and PPARγ gene expression in Skp2^−/− MEFs. Our data suggest that Skp2 plays an essential role in adipogenesis in MEFs in a manner that is at least in part independent of regulation of p27^Kip1 expression.     

胚胎干细胞周期调控的研究进展

胚胎干细胞（embryonic stem cells,Escs）具有自我复制和多潜能分化的特性。相对于体细胞,胚胎干细胞的细胞周期调控非常特别。比如,G1期较短;P53、RB等调控细胞周期“检验点”的蛋白分子功能“异常”等。胚胎干细胞细胞周期调控的研究对于研究胚胎干细胞的自我复制和多潜能性,都具有重要指导意义。该文将重点比较体细胞和胚胎干细胞在细胞周期调控方面的差异,并对近年来有关小鼠和人胚胎干细胞的细胞周期调控的研究进展进行介绍。     

p27Kip1 is required to maintain proliferative quiescence in the adult cochlea and pituitary

Cell cycle inhibitors, such as the cyclin-dependent kinase (Cdk) inhibitor proteins and retinoblastoma (Rb) family members, control exit from the cell cycle during the development of a variety of terminally differentiated tissues. It is unclear whether sustained expression of these proteins is required to prevent cell cycle re-entry in quiescent and terminally differentiated cells. The organ of Corti (cochlear sensory epithelium) and pars intermedia (intermediate lobe of the pituitary) are two tissues that share the characteristic of ongoing cell division in mice lacking either the p27^Kip1 Cdk inhibitor, Ink4 proteins, or Rb. Here, we use tamoxifen-inducible mouse models to delete p27^Kip1 in postnatal animals and show this is sufficient to induce proliferation in both the organ of Corti and pars intermedia. Thus, these tissues remain sensitive to the presence of p27^Kip1 even after their developmental exit from the cell cycle. The neonatal cochlea displayed heightened sensitivity to changes in p27^Kip1 expression, with a proliferative response higher than that of constitutive null mice. In adults, the proliferative response was reduced but was accompanied by increased cell survival. In contrast, re-establishment of normal p27^Kip1 expression in animals with established pituitary tumors, in an inducible “knock-on” model, led to cessation of pituitary tumor growth, indicating the cells had maintained their susceptibility to p27-mediated growth suppression. Although restoration of p27^Kip1 did not induce apoptosis, it did lead to resolution of pathological features and normalization of gene expression. Our data underscore the importance of p27^Kip1 expression in the maintenance of cellular quiescence and terminal differentiation.     

PDK1 regulates cell proliferation and cell cycle progression through control of cyclin D1 and p27Kip1 expression

PDK1 (3-phosphoinositide-dependent protein kinase 1) is a key mediator of signaling by phosphoinositide 3-kinase. To gain insight into the physiological importance of PDK1 in cell proliferation and cell cycle control, we established immortalized mouse embryonic fibroblasts (MEFs) from mice homozygous for a "floxed" allele of Pdk1 and from wild-type mice. Introduction of Cre recombinase by retrovirus-mediated gene transfer resulted in the depletion of PDK1 in Pdk1(lox/lox) MEFs but not in Pdk1(+/+) MEFs. The insulin-like growth factor-1-induced phosphorylation of various downstream effectors of PDK1, including Akt, glycogen synthase kinase 3, ribosomal protein S6, and p70 S6 kinase, was markedly inhibited in the PDK1-depleted (Pdk1-KO) MEFs. The rate of serum-induced cell proliferation was reduced; progression of the cell cycle from the G(0)-G(1) phase to the S phase was delayed, and cell cycle progression at G(2)-M phase was impaired in Pdk1-KO MEFs. These cells also manifested an increased level of p27(Kip1) expression and a reduced level of cyclin D1 expression during cell cycle progression. The defect in cell cycle progression from the G(0)-G(1) to the S phase in Pdk1-KO MEFs was rescued by forced expression of cyclin D1, whereas rescue of the defect in G(2)-M progression in these cells required both overexpression of cyclin D1 and depletion of p27(Kip1) by RNA interference. These data indicate that PDK1 plays an important role in cell proliferation and cell cycle progression by controlling the expression of both cyclin D1 and p27(Kip1).     

Regulation of p27Kip1 during gentamicin mediated hair cell death

The INK4 and Kip/Cip families of Cyclin Dependent Kinase inhibitors (CKIs) are regulators of the cell cycle. In addition, CKIS including p27^Kip1can protect cells from apoptosis in vitro. However, little is known about protective effect of p27^Kip1in vivo. We used systemic treatment with aminoglycosides to induce hair-cell death in the basilar papilla (BP), the auditory organ of the avian inner ear, and characterised the expression of p27^Kip1with confocal and immunofluorescence microscopy. In contrast to the adult mammalian cochlea where p27^Kip1is expressed only in supporting cells, p27^Kip1is found in the nuclei of both hair cells and supporting cells in the BP of the normal, mature bird. Forty-eight hours after gentamicin treatment, hair cells with TUNEL positive nuclei and hair cells with pyknotic nuclei were both detected, suggesting many hair cells die by apoptosis. When the BP was double labelled for p27^Kip1and myosin VIIa, a hair-cell specific protein, all dying hair cells that had been ejected from the epithelium were found to be myosin VIIa positive but negative for p27^Kip1even though nuclear remnants were still visible. In the transition zone where partial hair-cell loss occurs, freshly ejected hair cells lying immediately above the surface of the BP no longer expressed p27^Kip1. Damaged hair cells within the epithelium in the transition zone contained p27^Kip1in their cytoplasm but not in their nuclei. These data support recent in vitro findings suggesting that p27^Kip1protects cells from apoptosis and that its downregulation may be a general feature of programmed cell death.