p57kip2 regulates glial fate decision in adult neural stem cells

Our recent studies revealed p57kip2 as an intrinsic regulator of late gliogenesis and demonstrated that in oligodendroglial precursor cells p57kip2 inhibition leads to accelerated maturation. Adult neural stem cells have been described as a source of glial progenitors; however, the underlying mechanisms of cell fate specification are still poorly understood. Here, we have investigated whether p57kip2 can influence early events of glial determination and differentiation. We found that Sox2/GFAP double-positive cells express p57kip2 in stem cell niches of the adult brain. Short-hairpin RNA-mediated suppression of p57kip2 in cultured adult neural stem cells was found to strongly reduce astroglial characteristics, while oligodendroglial precursor features were increased. Importantly, this anti-astrogenic effect of p57kip2 suppression dominated the bone morphogenetic protein-mediated promotion of astroglial differentiation. Moreover, we observed that in p57kip2 knockdown cells, the BMP antagonist chordin was induced. Finally, when p57kip2-suppressed stem cells were transplanted into the adult spinal cord, fewer GFAP-positive cells were generated and oligodendroglial markers were induced when compared with control cells, demonstrating an effect of in vivo relevance.     

Opposing roles for the cyclin-dependent kinase inhibitor p27kip1 in the control of CD4+ T cell proliferation and effector function

Cell division drives T cell clonal expansion and differentiation, and is the result of concerted signaling from Ag, costimulatory, and growth factor receptors. How these mitogenic signals are coupled to the cell cycle machinery in primary T cells is not clear. We have focused on the role of p27kip1, a major cyclin-dependent kinase binding protein expressed by CD4+ T cells. Our studies using p27kip1 gene dosage demonstrate that early after activation, p27kip1 acts to promote, rather than inhibit, G1 to S phase progression within the first division cycle. However, throughout subsequent cell divisions p27kip1 behaves as a negative regulator, directly establishing the threshold amount of growth factor signaling required to support continued cell division. During this phase, signals from CD28 and IL-2R cooperate with the TCR to "tune" this threshold by inducing the degradation of p27kip1 protein, and we show that agents that block these pathways require elevated p27kip1 levels for their full antiproliferative activity. Finally, we show that p27kip1 opposes the development of CD4+ T cell effector function, and is required for the full development of anergy in response to a tolerizing stimulus. Our results suggest that p27kip1 plays a complex and important role in the regulation of cell division and effector function in primary CD4+ T cells.     

Regulated expression of cofilin and the consequent regulation of p27kip1 are essential for G1 phase progression

Cofilin, a ubiquitously expressed actin binding protein, is responsible for the formation of the actin cytoskeleton and is indispensable for cell cycle control. However, the association between cofilin expression and the cell cycle remains to be elucidated. In this study, we found that the expression level of cofilin up-regulated in G1 phase-arrested confluent cells, while knockdown of cofilin expression by small interference RNA (siRNA) in these cells led to a reduction in the population of G1 cells. To investigate the role of cofilin in the control of G1 phase progression, a tet-on gene expression system was introduced to over-express different concentrations of cofilin in cells. The results showed that G1 phase progression was blocked following induction of exogenous cofilin. A survey of the cell cycle proteins controlling the G1 phase progression revealed that the cyclin-dependent kinase inhibitor (CKI) p27kip1 was the primary molecule induced by over-expressed cofilin in a time and dose dependent manner. Up-regulated p27kip1 repressed phosphorylation of the retinoblastoma protein (Rb) mediated by cyclin D1/CDK4 activity. Conversely, siRNA against p27kip1 expression in the cofilin over-expressing cells released the G1 phase arrest. Furthermore, we found that over-expression of cofilin led to induction of p27kip1 gene promoter transactivation using luciferase reporter gene assay. This effect was associated with increase of p27kip1 mRNA transiently. In addition, inhibition of threonine-187 phosphorylation of p27kip1 protein for ubiquitinyl-proteasomal mediated degradation was also involved in up-regulation of p27 kip1. These data suggest that cofilin expression and its regulation of p27kip1 expression is important for the control of G1 phase progression.     

The nucleolar GTP-binding proteins Gnl2 and nucleostemin are required for retinal neurogenesis in developing zebrafish

Nucleostemin (NS), a member of a family of nucleolar GTP-binding proteins, is highly expressed in proliferating cells such as stem and cancer cells and is involved in the control of cell cycle progression. Both depletion and overexpression of NS result in stabilization of the tumor suppressor p53 protein in vitro. Although it has been previously suggested that NS has p53-independent functions, these to date remain unknown. Here, we report two zebrafish mutants recovered from forward and reverse genetic screens that carry loss of function mutations in two members of this nucleolar protein family, Guanine nucleotide binding-protein-like 2 (Gnl2) and Gnl3/NS. We demonstrate that these proteins are required for correct timing of cell cycle exit and subsequent neural differentiation in the brain and retina. Concomitantly, we observe aberrant expression of the cell cycle regulators cyclinD1 and p57kip2. Our models demonstrate that the loss of Gnl2 or NS induces p53 stabilization and p53-mediated apoptosis. However, the retinal differentiation defects are independent of p53 activation. Furthermore, this work demonstrates that Gnl2 and NS have both non-cell autonomously and cell-autonomous function in correct timing of cell cycle exit and neural differentiation. Finally, the data suggest that Gnl2 and NS affect cell cycle exit of neural progenitors by regulating the expression of cell cycle regulators independently of p53.     

Downregulation of p57kip2 promotes cell invasion via LIMK/cofilin pathway in human nasopharyngeal carcinoma cells

The members of Rho family are well known for their regulation of actin cytoskeleton to control cell migration. The Cip/kip members of cyclin‐dependent (CDK) inhibitors have shown to implicate in cell migration and cytoskeletal dynamics. p57^kip2, a CDK inhibitor, is frequently down‐regulated in several malignancy tumors. However, its biological roles in human nasopharyngeal carcinoma (NPC) cells remained to be investigated. Here, we found p57^kip2 has nuclear and cytoplasm distributions and depletion of endogenous p57^kip2 did not change the cell‐cycle progression. Inhibition of cell proliferation by mitomycin C promoted FBS‐mediated cell migration and accompanied with the downregulation of ΔNp63α and p57^kip2, but did not change the level of p27^kip1, another CDK inhibitor. By using siRNA transfection and cell migration/invasion assays, we found that knockdown of p57^kip2, but not ΔNp63α, involved in promotion of NPC cell migration and invasion via decrease of phospho‐cofilin (p‐cofilin). Treatment with Y‐27632, a specific ROCK inhibitor, we found that dysregulation of ROCK/cofilin pathway decreased p‐cofilin expression and induced cell migration. This change of p‐cofilin induced actin remodeling and pronounced increase of membrane protrusions. Further, silence of p57^kip2 not only decreased the interaction between p57^kip2 and LIMK‐1 assayed by immunoprecipitation but also reduced the level of phospho‐LIMK1/2. Therefore, this study indicated that dysregulation of p57^kip2 promoted cell migration and invasion through modulation of LIMK/cofilin signaling and suggested this induction of inappropriate cell motility might contribute to promoting tumor cell for metastasis. J. Cell. Biochem. 112: 3459–3468, 2011. © 2011 Wiley Periodicals, Inc.     

Dynamic temporal and spatial regulation of the cdk inhibitor p57(kip2) during embryo morphogenesis.

The complete developmental expression pattern of the cyclin dependent kinase inhibitor (CDKI) p57(kip2) has not been reported, here we report a detailed study of the localization of p57(kip2) protein during mouse organogenesis. We show that p57(kip2) is coincident with key stages of differentiation of several organs, some but not all of which are affected in Beckwith-Weidermann syndrome, a human congenital syndrome characterized by foetal overgrowth and childhood tumours.     

Cellular thiol status-dependent inhibition of tumor cell growth via modulation of p27<Superscript>kip1</Superscript> translocation and retinoblastoma protein phosphorylation by 1′-acetoxychavicol acetate

Summary. 1′-Acetoxychavicol acetate (ACA) has been shown to inhibit tumor cell growth, but there is limited information on its effects on cell signaling and the cell cycle control pathway. In this study, we sought to determine how ACA alters cell cycle and its related control factors in its growth inhibitory effect in Ehrlich ascites tumor cells (EATC). ACA caused an accumulation of cells in the G1 phase and an inhibition of DNA synthesis, which were reversed by supplementation with N-acetylcysteine (NAC) or glutathione ethyl ester (GEE). Furthermore, ACA decreased hyperphosphorylated Rb levels and increased hypophosphorylated Rb levels. NAC and GEE also abolished the decease in Rb phosphorylation by ACA. As Rb phosphorylation is regulated by G1 cyclin dependent kinase and CDK inhibitor p27^kip1, which is an important regulator of the mammalian cell cycle, we estimated the amount of p27^kip1 levels by western blotting. Treatment with ACA had virtually no effect on the amount of p27^kip1 levels, but caused a decrease in phosphorylated p27^kip1 and an increase in unphosphorylated p27^kip1 as well as an increase in the nuclear localization of p27^kip1. These events were abolished in the presence of NAC or GEE. These results suggest that in EATC, cell growth inhibition elicited by ACA involves decreases in Rb and p27^kip1 phosphorylation and an increase in nuclear localization of p27^kip1, and these events are dependent on the cellular thiol status.     

p57(Kip2) is degraded through the proteasome in osteoblasts stimulated to proliferation by transforming growth factor beta1

Cyclin-dependent kinase inhibitory proteins are negative regulators of the cell cycle. Although all the cyclin-dependent kinase inhibitory proteins may be involved in cell cycle control during a differentiation process, only p57(Kip2) is shown to be essential for embryonic development. However, the role of p57 in the control of the cell cycle is poorly understood. Using osteoblasts derived from the calvaria of rat fetus, we show that p57 is accumulated in cells starved by low serum. Cyclin-dependent kinase 2 activity was suppressed in these cells with a significant amount bound to p57. Treatment of the cells with transforming growth factor beta1 dramatically reduced the amount of p57, resulting in an activation of cyclin-dependent kinase 2 activity and the stimulation of cell proliferation. The decrease in p57 was inhibited by treating the cells with proteasome inhibitors, Z-Leu-Leu-Leu-aldehyde or lactacystin, but not with Z-Leu-Leu-aldehyde, which is an inhibitor of calpain, indicating that p57 is degraded through the proteasome pathway. p57 was also shown to be ubiquitinated in vitro. Because transforming growth factor beta1 not only stimulates the growth but also inhibits the differentiation of the cells in this system, our results may suggest a possible involvement of p57 in the control of osteoblastic cell proliferation and differentiation.     

The Drosophila F-box protein dSkp2 regulates cell proliferation by targeting Dacapo for degradation

Cell cycle progression is controlled by a complex regulatory network consisting of interacting positive and negative factors. In humans, the positive regulator Skp2, an F-box protein, has been a subject of intense investigation in part because of its oncogenic activity. By contrast, the molecular and developmental functions of its Drosophila homologue, dSkp2, are poorly understood. Here we investigate the role of dSkp2 by focusing on its functional relationship with Dacapo (Dap), the Drosophila homologue of the cyclin-dependent kinase inhibitors p21^cip1/p27^kip1/p57^kip2. We show that dSkp2 interacts physically with Dap and has a role in targeting Dap for ubiquitination and proteasome-mediated degradation. We present evidence that dSkp2 regulates cell cycle progression by antagonizing Dap in vivo. dSkp2 knockdown reduces cell density in the wing by prolonging the cell doubling time. In addition, the wing phenotype caused by dSkp2 knockdown resembles that caused by dap overexpression and can be partially suppressed by reducing the gene dose of dap. Our study thus documents a conserved functional relationship between dSkp2 and Dap in their control of cell cycle progression, suggesting the possibility of using Drosophila as a model system to study Skp2-mediated tumorigenesis.     

Ciglitazone-induced cellular anti-proliferation increases p27kip1 protein levels through both increased transcriptional activity and inhibition of proteasome degradation

While it is well established that PPARgamma ligands inhibit cell growth and induce apoptosis in colon cancer cells, the mechanism of these effects of PPARgamma ligands is unclear. In this report, we demonstrate that the PPARgamma ligand, ciglitazone, exhibits an anti-proliferative effect and blocks G1/S cell cycle progression through regulation of p27kip1 protein levels and inhibition of Cdk2 activity in HT-29 colon cancer cells. The ciglitazone-induced G1/S cell cycle arrest was noted only after 72 h of exposure, corresponding to elevated protein levels of p27kip1. However, an increase in p27kip1 protein synthesis as evidenced by increased p27kip1 gene promoter activity and mRNA abundance was observed as early as 24 h after exposure to ciglitazone. Proteasome activity, an additional mechanism of p27kip1 regulation, was dramatically inhibited after ciglitazone exposure, but only after 72 h of exposure. We also note that the effects of ciglitazone on p27kip1 gene regulation are PPRE independent. These data suggest that ciglitazone-induced G1/S arrest is through Cdk2 inhibition and an increase of p27kip1 protein levels which in turn is due a balance of ciglitazone's affect on new protein synthesis and degradation.     

Pirh2

Ubiquitylation is currently recognized as a major posttranslational modification that regulates diverse cellular processes. Pirh2 is a ubiquitin E3 ligase that regulates the turnover and functionality of several proteins involved in cell proliferation and differentiation, cell cycle checkpoints, and cell death. Here we review the role of Pirh2 as a regulator of the DNA damage response through the ubiquitylation of p53, Chk2, p73, and PolH. By ubiquitylating these proteins, Pirh2 regulates cell cycle checkpoints and cell death in response to DNA double-strand breaks or the formation of bulky DNA lesions. We also discuss how Pirh2 affects cell proliferation and differentiation in unstressed conditions through ubiquitylation and degradation of c-Myc, p63, and p27^kip1. Finally, we link these different functions of Pirh2 to its role as a tumor suppressor in mice and as a prognosis marker in various human cancer subtypes.     

Thyroid cell proliferation in response to forced expression of gap junction proteins

Gap junctions are known to play a role in the control of cell proliferation, and connexins (Cx) are considered to be tumor suppressors. However, the effects of Cx on cell proliferation are dependent on the Cx which is expressed and on the cell type under consideration. We previously found that restoration of cell-to-cell communication by stable transfection of two independent thyroid-derived cell lines, FRTL-5 and FRT cells, with the Cx32 gene induced a marked reduction of their proliferation rate. This study aimed i) at determining whether Cx43, which is coexpressed with Cx32 by thyroid epithelial cells, exerts the same action as Cx32 on cell proliferation and ii) at identifying alterations of the cell cycle control system that might account for the Cx32-induced proliferation slowdown in thyrocytes. In contrast with previous data on different epithelial cell types, we report that restoration of intercellular communication in FRTL-5 and FRT cells by stable expression of Cx43 did not modify their proliferation properties. Cell cycle analyses revealed that the Cx32-induced proliferation slow-down was related to a lengthening of the G1 phase. The level of expression of two regulatory proteins of the Cip/Kip cyclin-dependent kinase inhibitor family, p27kip1 and p2cip1, was increased in the two cell lines expressing Cx32. In conclusion, Cx32 and Cx43, physiologically coexpressed by thyrocytes, have a differential impact on thyroid cell proliferation in vitro. The cyclin-dependent kinase inhibitors, p27kip1 and p21cip1 might represent cell cycle effectors relaying the down-regulatory effect of Cx32 on the proliferation of thyroid epithelial cells.