Mounting evidence indicates cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family, including p57Kip2 and p27Kip1, control not only cell cycle exit but also corticogenesis. Nevertheless, distinct activities of p57Kip2 remain poorly defined. Using in vivo and culture approaches, we show p57Kip2 overexpression at E14.5–15.5 elicits precursor cell cycle exit, promotes transition from proliferation to neuronal differentiation, and enhances process outgrowth, while opposite effects occur in p57Kip2-deficient precursors. Studies at later ages indicate p57Kip2 overexpression also induces precocious glial differentiation, suggesting stage-dependent effects. In embryonic cortex, p57Kip2 overexpression advances cell radial migration and alters postnatal laminar positioning. While both CKIs induce differentiation, p57Kip2 was twice as effective as p27Kip1 in inducing neuronal differentiation and was not permissive to astrogliogenic effects of ciliary neurotrophic factor, suggesting that the CKIs differentially modulate cell fate decisions. At molecular levels, although highly conserved N-terminal regions of both CKIs elicit cycle withdrawal and differentiation, the C-terminal region of p57Kip2 alone inhibits in vivo migration. Furthermore, p57Kip2 effects on neurogenesis and gliogenesis require the N-terminal cyclin/CDK binding/inhibitory domains, while previous p27Kip1 studies report cell cycle-independent functions. These observations suggest p57Kip2 coordinates multiple stages of corticogenesis and exhibits distinct and common activities compared with related family member p27Kip1.
gliogenesis; in utero electroporation; neurite outgrowth; neurogenesis; transfection
The cyclin/cyclin-dependent kinase (cdk) inhibitor p27kip1 is thought to be responsible for the onset and maintenance of the quiescent state. It is possible, however, that cells respond differently to p27kip1 in different conditions, and using a BALB/c-3T3 cell line (termed p27-47) that inducibly expresses high levels of this protein, we show that the effect of p27kip1 on cell cycle traverse is determined by cell density. We found that ectopic expression of p27kip1 blocked the proliferation of p27-47 cells at high density but had little effect on the growth of cells at low density whether exponentially cycling or stimulated from quiescence. Regardless of cell density, the activities of cdk4 and cdk2 were markedly repressed by p27kip1 expression, as was the cdk4-dependent dissociation of E2F4/p130 complexes. Infection of cells with SV40, a DNA tumor virus known to abrogate formation of p130- and Rb-containing complexes, allowed dense cultures to proliferate in the presence of supraphysiological amounts of p27kip1 but did not stimulate cell cycle traverse when cultures were cotreated with the potent cdk2 inhibitor roscovitine. Our data suggest that residual levels of cyclin/cdk activity persist in p27kip1-expressing p27-47 cells and are sufficient for the growth of low-density cells and of high-density cells infected with SV40, and that effective disruption of p130 and/or Rb complexes is obligatory for the proliferation of high-density cultures.
BACKGROUND: Low levels of the cyclin-dependent kinase inhibitor p27Kip1 are associated with poor prognosis in cancer. It is unclear whether this is related strictly to p27Kip1-mediated cell cycle inhibition or to other, possibly extranuclear, roles of this protein. In this study, we examined p27Kip1 expression in quiescent and activated lymphocytes. T-cell membranes have been shown to possess sphingolipid and cholesterol-rich microdomains that are insoluble in non-ionic detergents. These "rafts" provide a scaffold for signaling proteins. Signal transduction coincides with coalescence of these microdomains into larger complexes. METHODS: Localization of p27Kip1 was studied by electron and confocal microscopy. Association of p27Kip1 with membrane microdomains in unstimulated and stimulated lymphocytes was determined using Western blots analysis of isolated membranes variably treated with detergents. RESULTS: We demonstrated that p27Kip1 was present in clusters associated with the plasma membrane in normal lymphocytes. The solubility profile of p27Kip1 in isolated membranes indicated that it was localized to raft structures. When lymphocytes were stimulated, however, p27Kip1 was excluded from aggregated raft complexes. CONCLUSIONS: This study identifies, for the first time, the localization of p27 within a membrane microdomain associated with signaling. Because some cell surface signaling complexes lose p27Kip1 upon cellular activation, p27Kip1 may play a functional role in modulating membrane signaling.
Vascular proliferative diseases are characterized by VSMC proliferation and migration. Kinase interacting with stathmin (KIS) targets 2 key regulators of cell proliferation and migration, the cyclin-dependent kinase inhibitor p27Kip1 and the microtubule-destabilizing protein stathmin. Phosphorylation of p27Kip1 by KIS leads to cell-cycle progression, whereas the target sequence and the physiological relevance of KIS-mediated stathmin phosphorylation in VSMCs are unknown. Here we demonstrated that vascular wound repair in KIS–/– mice resulted in accelerated formation of neointima, which is composed predominantly of VSMCs. Deletion of KIS increased VSMC migratory activity and cytoplasmic tubulin destabilizing activity, but abolished VSMC proliferation through the delayed nuclear export and degradation of p27Kip1. This promigratory phenotype resulted from increased stathmin protein levels, caused by a lack of KIS-mediated stathmin phosphorylation at serine 38 and diminished stathmin protein degradation. Downregulation of stathmin in KIS–/– VSMCs fully restored the phenotype, and stathmin-deficient mice demonstrated reduced lesion formation in response to vascular injury. These data suggest that KIS protects against excessive neointima formation by opposing stathmin-mediated VSMC migration and that VSMC migration represents a major mechanism of vascular wound repair, constituting a relevant target and mechanism for therapeutic interventions.
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.
Neuronal progenitor cells of the anterior subventricular zone (SVZa) migrate along the rostral migratory stream (RMS) to the olfactory bulb, where they exit the cell cycle and differentiate. The molecular mechanisms that regulate SVZa progenitor proliferation and cell cycle exit are largely undefined. We investigated the role of p27KIP1 in regulating cell proliferation and survival in the RMS and olfactory bulb between postnatal day 1 (P1) and P14, the peak period of olfactory bulb neuron generation. A large proportion of cells in the RMS and the olfactory bulb express cytoplasmic p27KIP1, but a small percentage display high nuclear p27KIP1 immunostaining, which exhibit a caudallow-rostralhigh gradient: lowest in the SVZa and highest in the glomerular layer of the olfactory bulb. p27KIP1 is also present in the nucleus and/or the cytoplasm of neuron-specific type III β-tubulin(+) cells. Cells with strong nuclear p27KIP1 expression are BrdU(−) and Ki67(−). The percentage of BrdU(+) cells in the SVZa, RMS and olfactory bulb is higher in p27KIP1 null than wild type (WT) mice at all ages analyzed. Consistent with these findings, p27KIP1 overexpression in cultured p27KIP1 null and WT SVZ cells reduced cell proliferation and self-renewal. Finally, in p27KIP1 null mice, the diameter of the horizontal limb of the RMS is larger than in WT mice, and development of the olfactory bulb granule cell layer is delayed, together with increased apoptotic cell density. Our results indicate that in the postnatal brain p27KIP1 regulates the proliferation and survival of neuronal cells in the RMS and olfactory bulb.
cell differentiation; cell proliferation; cell survival; cyclin dependent kinase inhibitor; neuronal progenitor cells; subventricular zone
We report that cyclin D3/cdk4 kinase activity is regulated by p27kip1 in BALB/c 3T3 cells. The association of p27kip1 was found to result in inhibition of cyclin D3 activity as measured by immune complex kinase assays utilizing cyclin D3-specific antibodies. The ternary p27kip1/cyclin D3/cdk4 complexes do exhibit kinase activity when measured in immune complex kinase assays utilizing p27kip1-specific antibodies. The association of p27kip1 with cyclin D3 was highest in quiescent cells and declined upon mitogenic stimulation, concomitantly with declines in the total level of p27kip1 protein. The decline in this association could be elicited by PDGF treatment alone; this was not sufficient, however, for activation of cyclin D3 activity, which also required the presence of factors in platelet-poor plasma in the culturing medium. Unlike cyclin D3 activity, which was detected only in growing cells, p27kip1 kinase activity was present throughout the cell cycle. Since we found that the p27kip1 activity was dependent on cyclin D3 and cdk4, we compared the substrate specificity of the active ternary complex containing p27kip1 and the active cyclin D3 lacking p27kip1 by tryptic phosphopeptide mapping of GST-Rb phosphorylated in vitro and also by comparing the relative phosphorylation activity toward a panel of peptide substrates. We found that ternary p27kip1/cyclin D3/cdk4 complexes exhibited a different specificity than the active binary cyclin D3/cdk4 complexes, suggesting that p27kip1 has the capacity to both inhibit cyclin D/cdk4 activity as well as to modulate cyclin D3/cdk4 activity by altering its substrate preference.
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.
Cyclin-Dependent Kinase Inhibitor p57Kip2; Embryonic Development/physiology; Nervous System/cytology/*embryology; Brain/embryology; Neuronal Differentiation
p27Kip1 is a member of the Cip-Kip family of cyclin-dependent kinase (Cdk) inhibitors that binds to cyclin-Cdk complexes and inhibits their catalytic activity in response to antiproliferative stimuli. p27Kip1 is regulated by several posttranscriptional mechanisms, including subcellular localization. We have identified a component of the nuclear pore complex (NPC), termed Nup50, through its two-hybrid interactions with p27Kip1. Nup50 is a nucleoplasmically oriented component of the nuclear pore complex with a role in protein export (T. Guan, R. H. Kehlenbach, E. C. Schirmer, A. Kehlenbach, F. Fan, B. E. Clurman, N. Arnheim, and L. Gerace, Mol. Cell. Biol. 20:5619–5630, 2000). We found that murine Nup50 is a widely expressed nucleoporin and that Nup50 expression is highest in the developing neural tube and adult testes. We have also examined interactions between Nup50 and the NPC and found specific two-hybrid interactions between Nup50 and several well-defined components of the NPC, as well as coimmunoprecipitation of Nup50 with the nucleoporin Nup153 from transfected mammalian cells. In order to study Nup50 function in vivo, we cloned the mouse Nup50 genomic locus and created a targeted Nup50 deletion in the mouse germ line. Nup50 disruption resulted in a complex phenotype characterized by late embryonic lethality, neural tube defects, and intrauterine growth retardation. Although Nup50-null mouse embryo fibroblasts exhibited no defects in either cell cycle control or p27Kip1 regulation, Nup50 deletion was associated with abnormalities in p27Kip1 expression and cell proliferation in the developing neuroepithelium. We conclude that Nup50 is a nucleoporin with essential functions during mouse development.
p27Kip1 is a cyclin-dependent kinase inhibitor that regulates the G1/S transition. Increased degradation of p27Kip1 is associated with cellular transformation. Previous work demonstrated that the ubiquitin ligases KPC1/KPC2 and SCFSkp2 ubiquitinate p27Kip1 in G1 and early S, respectively. The regulation of these ligases remains unclear. We report here that the USP19 deubiquitinating enzyme interacts with and stabilizes KPC1, thereby modulating p27Kip1 levels and cell proliferation. Cells depleted of USP19 by RNA interference exhibited an inhibition of cell proliferation, progressing more slowly from G0/G1 to S phase, and accumulated p27Kip1. This increase in p27Kip1 was associated with normal levels of Skp2 but reduced levels of KPC1. The overexpression of KPC1 or the use of p27−/− cells inhibited significantly the growth defect observed upon USP19 depletion. KPC1 was ubiquitinated in vivo and stabilized by proteasome inhibitors and by overexpression of USP19, and it also coimmunoprecipitated with USP19. Our results identify USP19 as the first deubiquitinating enzyme that regulates the stability of a cyclin-dependent kinase inhibitor and demonstrate that progression through G1 to S phase is, like the metaphase-anaphase transition, controlled in a hierarchical, multilayered fashion.
The p27Kip1 protein plays a critical role in the regulation of cell proliferation through the inhibition of cyclin-dependent kinase activity. Translation of p27Kip1 is directed by an internal ribosomal entry site (IRES) in the 5′ nontranslated region of p27Kip1 mRNA. Here, we report that polypyrimidine tract-binding protein (PTB) specifically enhances the IRES activity of p27Kip1 mRNA through an interaction with the IRES element. We found that addition of PTB to an in vitro translation system and overexpression of PTB in 293T cells augmented the IRES activity of p27Kip1 mRNA but that knockdown of PTB by introduction of PTB-specific small interfering RNAs (siRNAs) diminished the IRES activity of p27Kip1 mRNA. Moreover, the G1 phase in the cell cycle (which is maintained in part by p27Kip1) was shortened in cells depleted of PTB by siRNA knockdown. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced differentiation in HL60 cells was used to examine PTB-induced modulation of p27Kip1 protein synthesis during differentiation. The IRES activity of p27Kip1 mRNA in HL60 cells was increased by TPA treatment (with a concomitant increase in PTB protein levels), but the levels of p27Kip1 mRNA remained unchanged. Together, these data suggest that PTB modulates cell cycle and differentiation, at least in part, by enhancing the IRES activity of p27Kip1 mRNA.
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.
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.
Background/Aims: Overexpression of the G1 cyclins, D1 and E, and/or downregulation of p27Kip1 allow uncontrolled tumour cell proliferation. This study investigated the relation between these three cell cycle proteins and tumour proliferation in bladder cancer.
Method: Nuclear expression of cyclin D1, cyclin E, and p27Kip1 was determined immunohistochemically in 52 primary transitional cell carcinomas, and the Ki-67 proliferation marker was also assessed. For each protein, the percentage of positive tumour cell nuclei was determined and analysed as a continuous variable.
Results: Advancing tumour grade and pathological stage were accompanied by increasing proliferation indices, but decreasing p27Kip1 and cyclin D1 expression, with no significant change in cyclin E expression. Overall, cyclin D1 and E expression did not correlate with proliferation. However, in cyclin D1 overexpressing tumours (⩾ 5% nuclei positive), the level of cyclin D1 expression positively correlated with proliferation. The correlation between cyclin E expression and proliferation changed from positive to negative with increasing levels of cyclin E expression, accompanied by a coordinate increase in p27Kip1 expression. Overall, there was an inverse association between p27Kip1 expression and proliferation. However, a subset of tumours displayed high proliferation indices despite high p27Kip1 expression. The G1 cyclin index (sum of the level of expression of cyclins D1 and E) correlated positively with proliferation in superficial but not muscle invasive tumours. This correlation was stronger when the G1 cyclin index was adjusted for p27Kip1 expression.
Conclusion: These findings support a role for these proteins in the proliferation, differentiation, and progression of bladder transitional cell carcinomas.
cell cycle; bladder cancer; cyclin; proliferation
K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.
The universal cyclin-Cdk inhibitor p27Kip1 functions as a tumor suppressor and reduced levels of p27Kip1 connote poor prognosis in several human malignancies. p27Kip1 levels are predominately regulated by ubiquitin-mediated turnover of the protein, which is marked for destruction by the E3 ubiquitin ligase SCFSkp2 complex following its phosphorylation by the cyclin E-Cdk2 complex. Binding of phospho-p27Kip1 is directed by the Skp2 F-box protein, and this is greatly augmented by its allosteric regulator Cks1. We have established that programmed expression of c-Myc in the B cells of Eμ-Myc transgenic mice triggers p27Kip1 destruction by inducing Cks1, that this response controls Myc-driven proliferation, and that loss of Cks1 markedly delays Myc-induced lymphomagenesis and cancels the dissemination of these tumors. Here, we report that elevated levels of Skp2 are a characteristic of Eμ-Myc lymphomas and of human Burkitt lymphoma that bear MYC/immunoglobulin chromosomal translocations. As expected, Myc-mediated suppression of p27Kip1 was abolished in Skp2-null Eμ-Myc B cells. However, the impact of Skp2 loss on Myc-driven proliferation and lymphomagenesis was surprisingly modest compared to the effects of Cks1 loss. Collectively these findings suggest that Cks1 targets in addition to p27Kip1 are critical for Myc-driven proliferation and tumorigenesis.
Myc; Skp2; p27Kip1; lymphomagenesis
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.
Cell cycle inhibitors, such as the cyclin-dependent kinase (Cdk) inhibitor proteins and retinoblastoma (Rb) family members, control exit from the cell cycle during the development of a variety of terminally differentiated tissues. It is unclear whether sustained expression of these proteins is required to prevent cell cycle re-entry in quiescent and terminally differentiated cells. The organ of Corti (cochlear sensory epithelium) and pars intermedia (intermediate lobe of the pituitary) are two tissues that share the characteristic of ongoing cell division in mice lacking either the p27Kip1 Cdk inhibitor, Ink4 proteins or Rb. Here, we use tamoxifen-inducible mouse models to delete p27Kip1 in postnatal animals and show this is sufficient to induce proliferation in both the organ of Corti and pars intermedia. Thus, these tissues remain sensitive to the presence of p27Kip1 even after their developmental exit from the cell cycle. The neonatal cochlea displayed heightened sensitivity to changes in p27Kip1 expression, with a proliferative response higher than that of constitutive null mice. In adults, the proliferative response was reduced but was accompanied by increased cell survival. In contrast, re-establishment of normal p27Kip1 expression in animals with established pituitary tumors, in an inducible “knock-on” model, led to cessation of pituitary tumor growth, indicating the cells had maintained their susceptibility to p27-mediated growth suppression. Although restoration of p27Kip1 did not induce apoptosis, it did lead to resolution of pathological features and normalization of gene expression. Our data underscore the importance of p27Kip1 expression in the maintenance of cellular quiescence and terminal differentiation.
proliferation; cell cycle; p27; Cdk inhibitor; auditory; cochlea; pituitary
p27kip1 is a cyclin-dependent kinase inhibitor that regulates progression from G1 into S phase. Aberrations in cell cycle control are often observed in tumors and might even be necessary in tumor development. Recent reports showed that low p27kip1 expression is associated with poor prognosis in several tumors and leukemia. To investigate the expression of p27kip1 in malignant lymphomas and elucidate the role of p27kip1 as a possible prognostic indicator, the authors performed an immunohistochemical staining of p27kip1 correlated with Ki-67 labelling index and clinical parameters. p27kip1 expression was reduced variably in most malignant lymphomas and inversely correlated with Ki-67 labelling index (p=0.0151). Regarding chemotherapeutic response, p271kip1 expression in the complete remission group showed statistically significant difference in expression compared to the progressive disease group (p=0.0021). There were significant differences in survival between cases with low and high p27kip1 expression (p=0.0071). In a multivariate Cox analysis, p27kip1 expression was independent prognostic factors as well as other known prognostic factors including age, grade, stage and chemotherapeutic response. In conclusion, the study suggests that reduced expression of p27kip1 protein may play a role in the pathogenesis and biologically aggressive behavior of malignant lymphomas.
Interleukin (IL)-7 is required for survival and homeostatic proliferation of T lymphocytes. The survival effect of IL-7 is primarily through regulation of Bcl-2 family members; however, the proliferative mechanism is unclear. It has not been determined whether the IL-7 receptor actually delivers a proliferative signal or whether, by promoting survival, proliferation results from signals other than the IL-7 receptor. We show that in an IL-7–dependent T cell line, cells protected from apoptosis nevertheless underwent cell cycle arrest after IL-7 withdrawal. This arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27Kip1 through a posttranslational mechanism. Overexpression of p27Kip1 induced G1 arrest in the presence of IL-7, whereas knockdown of p27Kip1 by small interfering RNA promoted S phase entry after IL-7 withdrawal. CD4 or CD8 T cells transferred into IL-7–deficient hosts underwent G1 arrest, whereas 27Kip1-deficient T cells underwent proliferation. We observed that IL-7 withdrawal activated protein kinase C (PKC)θ and that inhibition of PKCθ with a pharmacological inhibitor completely blocked the rise of p27Kip1 and rescued cells from G1 arrest. The conventional pathway to breakdown of p27Kip1 is mediated by S phase kinase-associated protein 2; however, our evidence suggests that PKCθ acts via a distinct, unknown pathway inducing G1 arrest after IL-7 withdrawal from T cells. Hence, IL-7 maintains T cell proliferation through a novel pathway of p27Kip1 regulation.
Timely cell cycle regulation is conducted by sequential activation of a family of serine-threonine kinases called cycle dependent kinases (CDKs). Tight CDK regulation involves cyclin dependent kinase inhibitors (CKIs) which ensure the correct timing of CDK activation in different phases of the cell cycle. One CKI of importance is p27KIP1. The regulation and cellular localization of p27KIP1 can result in biologically contradicting roles when found in the nucleus or cytoplasm of both normal and tumor cells. The p27KIP1 protein is mainly regulated by proteasomal degradation and its downregulation is often correlated with poor prognosis in several types of human cancers. The protein can also be functionally inactivated by cytoplasmic localization or by phosphorylation. The p27KIP1 protein is an unconventional tumor suppressor because mutation of its gene is extremely rare in tumors, implying the normal function of the protein is deranged during tumor development. While the tumor suppressor function is mediated by p27KIP1's inhibitory interactions with the cyclin/CDK complexes, its oncogenic function is cyclin/CDK independent, and in many cases correlates with cytoplasmic localization. Here we review the basic features and novel aspects of the p27KIP1 protein, which displays genetically separable tumor suppressing and oncogenic functions.
cell cycle; cyclin-dependent kinase inhibitor p27; cyclin-dependent kinases; tumor suppressor proteins
CD8 T cells exhibit dynamic alterations in proliferation and apoptosis during various phases of the CD8 T cell response, but the mechanisms that regulate cellular proliferation from the standpoint of CD8 T cell memory are not well defined. The cyclin-dependent kinase inhibitor p27Kip1 functions as a negative regulator of the cell cycle in various cell types including T cells and it has been implicated in regulating cellular processes including differentiation, transcription and migration. Here, we investigated whether p27Kip1 regulates CD8 T cell memory by T cell-intrinsic or T cell-extrinsic mechanisms, by conditional ablation of p27Kip1 in T cells or non-T cells. Studies of T cell responses to an acute viral infection show that p27Kip1 negatively regulates the proliferation of CD8 T cells by T cell-intrinsic mechanisms. However, the enhanced proliferation of CD8 T cells induced by T cell-specific p27Kip1 deficiency minimally affects the primary expansion or the magnitude of CD8 T cell memory. Unexpectedly, p27Kip1 ablation in non-T cells markedly augmented the number of high quality memory CD8 T cells by enhancing the accumulation of memory precursor effector cells without increasing their proliferation. Further studies show that p27Kip1 deficiency in immunizing DCs fail to enhance CD8 T cell memory. Nevertheless, we have delineated the T cell-intrinsic, anti-proliferative activities of p27Kip1 in CD8 T cells from its role as a factor in non-T cells that restricts the development of CD8 T cell memory. These findings have implications in vaccine development and understanding the mechanisms that maintain T cell homeostasis.
CD8 T cells; Cell cycle; Memory; p27Kip1; Proliferation
Actin cytoskeleton remodeling is under the regulation of multiple proteins with various activities. Here, we demonstrate that the γ2 isoform of Casein Kinase I (CKIγ2) is part of a novel molecular path regulating the formation of actin stress fibers. We show that overexpression of CKIγ2 in fibroblasts alters cell morphology by impairing actin stress fibers formation. We demonstrate that this is concomitant with increased phosphorylation of the CDK inhibitor p27Kip and lower levels of activated RhoA, and is dependent on CKIγ2 catalytic activity. Moreover, we report that roscovitine, a potent inhibitor of cyclin-dependent kinases, including Cdk5, decreases p27Kip protein levels and restores actin stress fibers formation in CKIγ2 overexpressing cells, suggesting the existence of a CKIγ2-Cdk5-p27Kip-RhoA pathway in regulating actin remodeling. On the other hand, we also show that in a manner independent of its catalytic activity, CKIγ2 delays cell cycle progression through G1. Collectively our findings reveal that CKIγ2 is a novel player in the control of actin cytoskeleton dynamics and cell proliferation.
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.
It is widely acknowledged that cultured myoblasts can not differentiate at very low density. Here we analyzed the mechanism through which cell density influences myogenic differentiation in vitro. By comparing the behavior of C2C12 myoblasts at opposite cell densities, we found that, when cells are sparse, failure to undergo terminal differentiation is independent from cell cycle control and reflects the lack of p27Kip1 and MyoD in proliferating myoblasts. We show that inhibition of p27Kip1 expression impairs C2C12 cell differentiation at high density, while exogenous p27Kip1 allows low-density cultured C2C12 cells to enter the differentiative program by regulating MyoD levels in undifferentiated myoblasts. We also demonstrate that the early induction of p27Kip1 is a critical step of the N-cadherin-dependent signaling involved in myogenesis. Overall, our data support an active role of p27Kip1 in the decision of myoblasts to commit to terminal differentiation, distinct from the regulation of cell proliferation, and identify a pathway that, reasonably, operates in vivo during myogenesis and might be part of the phenomenon known as “community effect”.