The cyclin-dependent kinase inhibitors p27Kip1 and p21Cip1 cooperate to restrict proliferative life span in differentiating ovarian cells

The timing of cellular exit from the cell cycle during differentiation is specific for each cell type or lineage. Granulosa cells in the ovary establish quiescence within several hours after the ovulation-inducing luteinizing hormone surge, whereas they undergo differentiation into corpora lutea. The expression of Cdk inhibitors p21(Cip1/Waf1) and p27(Kip1) is up-regulated during this process, suggesting that these cell cycle inhibitors are involved in restricting proliferative capacity of differentiating granulosa cells. Here we demonstrate that the lack of p27(Kip1) and p21(Cip1) synergistically renders granulosa cells extended an proliferative life span. Immunohistochemical analyses demonstrated that corpora lutea of p27(Kip1), p21(Cip1) double-null mice showed large numbers of cells with bromodeoxyuridine incorporation and high proliferative cell nuclear antigen expression, which were more remarkable than those in p27(Kip1) single-deficient mice showing modest hyperproliferation. In contrast, differentiating granulosa cells in p21(Cip1)-deficient mice ceased proliferation similarly to those in wild-type mice. Interestingly, granulosa cells isolated from p27(Kip1), p21(Cip1) double-null mice exhibited markedly prolonged proliferative life span in culture, unlike cells with other genotypes. Cultured p27(Kip1), p21(Cip1) double-null granulosa cells maintained expression of steroidogenic enzymes and gonadotropin receptors through 8-10 passages and could undergo further differentiation in responses to cAMP accumulation. Thus, the cooperation of p27(Kip1) and p21(Cip1) is critical for withdrawal of granulosa cells from the cell cycle, in concert with luteal differentiation and possibly culture-induced senescence.     

A pro-apoptotic effect of the CDK inhibitor p57(Kip2) on staurosporine-induced apoptosis in HeLa cells

Apoptosis, or programmed cell death, is involved in many biological events, including tumorigenesis. Recently, it has been reported that two members of the Cip/Kip family of CDK inhibitors, p21(Cip1) and p27(Kip1), are involved in the regulation of apoptosis. Here, we report that selective expression of the third member in this family, p57(Kip2), potentiated staurosporine-induced apoptosis in HeLa cells. This pro-apoptotic effect was associated with an increased caspase-3 activity. In contrast, glucocorticoid treatment, despite inducing p57(Kip2) expression in HeLa cells, was found to have an inhibitory effect on staurosporine-induced apoptosis. This anti-apoptotic effect of glucocorticoids could be explained by a concomitant increase in Bcl-x(L) expression. The results presented in this study show that p57(Kip2) has a stimulatory effect on apoptosis induced by staurosporine, suggesting a role for p57(Kip2) in the response of tumor cells to cytotoxic drugs.     

P-TEFb- the final frontier

Cyclin-dependent kinases (CDKs) play a central role in the orderly transition from one phase of the eukaryotic mitotic cell division cycle to the next. In this context, p27^Kip1 (one of the CIP/KIP family of CDK specific inhibitors in mammals) or its functional analogue in other eukarya prevents a premature transition from G1 to S-phase. Recent studies have revealed that expression of a second member of this family, p57^Kip2, is induced as trophoblast stem (TS) cells differentiate into trophoblast giant (TG) cells. p57 then inhibits CDK1 activity, an enzyme essential for initiating mitosis, thereby triggering genome endoreduplication (multiple S-phases without an intervening mitosis). Expression of p21^Cip1, the third member of this family, is also induced in during differentiation of TS cells into TG cells where it appears to play a role in suppressing the DNA damage response pathway. Given the fact that p21 and p57 are unique to mammals, the question arises as to whether one or both of these proteins are responsible for the induction and maintenance of polyploidy during mammalian development.     

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.     

Cell cycle regulation during mouse olfactory neurogenesis.

The development of the nervous system requires a strict control of cell cycle entry and withdrawal. The olfactory epithelium (OE) is noticeable by its ability to yield new neurons not only during development but also continuously during adulthood. The aim of our study was to investigate, by biochemical and immunohistochemical methods, which cell cycle regulators are involved in the control of neuron production during OE development and maturity. At birth, olfactory neural progenitors, the basal cells, exhibited a high mitogenic and neurogenic activity, decreasing in the following weeks together with the drop in expression of several cell cycle regulators. p27Kip1 and p18Ink4c, at birth, were expressed in the whole basal cell layer, whereas p16Ink4a, p19Ink4d, and p21Cip1 were rather located in differentiating or mature neurons. CDK inhibitors may thus act sequentially during this developmental neurogenic process. By comparison, in the adult OE, in which most neural precursors were quiescent, these cells still exhibited p18Ink4c expression but only occasionally p27Kip1 expression. It suggests that p18Ink4c may contribute to maintain basal cells in a quiescent state, whereas p27Kip1 expression in these cells may be rather linked to their neurogenic activity, which declines with age. In keeping with this hypothesis, transgenic mice that lacked p27Kip1 expression displayed a higher rate of cell proliferation versus differentiation in their OE. In these mice, a down-regulation of positive cell cycle regulators was observed that may contribute to compensate for the absence of p27Kip1. Taken together, the present data suggest distinct functions for CDK inhibitors, either in the control of cell cycle exit and differentiation during neurogenesis (respectively, p27Kip1 and p19Ink4d) or in the maintenance of a quiescent state in neural progenitors (p18Ink4c) or neurons (p21Cip1) in adults.     

Age-dependent changes of p57(Kip2) and p21(Cip1/Waf1) expression in skeletal muscle and lung of mice

p57(Kip2) and p21(Cip1/Waf1) are members of cyclin-dependent kinase (Cdk) inhibitors which play critical roles in the terminal differentiation of skeletal muscle and lung. We investigated mRNA levels of p57(Kip2) and p21(Cip1/Waf1) in skeletal muscle and lung of mice during maturation and aging using Northern hybridization. The mRNA levels of p57(Kip2) and p21(Cip1/Waf1) decreased in skeletal muscle and lung of mice during maturation and aging except that the level of p21(Cip1/Waf1) mRNA in skeletal muscle of mice showed an increase only during maturation. The decrease of the p57(Kip2) mRNA level involved neither a change of DNA methylation at the promoter region nor an alteration of the imprinting status in aged mice. The decreases of p57(Kip2) and p21(Cip1/Waf1) mRNA levels during aging suggest that the process of tissue-specific terminal differentiation may be gradually downregulated with senescence in tissues where p57(Kip2) and p21(Cip1/Waf1) play key roles in differentiation. The downregulation of p57(Kip2) and p21(Cip1/Waf1) during aging is contrary to the upregulation of Cdk inhibitors during cellular replicative senescence, indicating that aging in an organismal level is mediated by mechanisms different from replicative senescence of cultured cells.     

Modulation of the expression of the Cip/Kip family of cyclin-dependent kinase inhibitors in foetal developing lungs of hamsters

We examine the cell proliferation activity and expression of cyclin-dependent kinase inhibitors of the Cip/Kip family, p21Cip1, p27Kip1 and p57Kip2, in foetal hamster lungs to determine the expression patterns of the cyclin-dependent kinase inhibitors and to clarify the relationship between expression of the cyclin-dependent kinase inhibitors and lung development. Foetal hamster lungs on gestational days 12.5-16 (the day of birth) and adult lungs were fixed in 4% paraformaldehyde. Frozen sections were immunostained for the cyclin-dependent kinase inhibitors, and examined by immunostaining for Ki-67 and bromodeoxyuridine to determine the proliferation activity of the foetal lungs. During the foetal period, cell proliferation activity, as analysed by Ki-67 or bromodeoxyuridine labelling, decreased with development of the lung. In contrast to the gradual decrease of cell proliferation activity, cells with p27Kip1 immunoreactivity increased with development. On the other hand, p21Cip1-positive cells were most prominent around gestational day 14.5, while after birth positive cells decreased markedly. A few p57Kip2-positive cells were detected in the bronchiolar epithelium on gestational day 14.5. Western blotting analyses confirmed these immunostaining patterns. Thus, the levels of the cyclin-dependent kinase inhibitors of the Cip/Kip family are modulated in the lungs during the foetal period, and each shows a unique expression pattern. The cyclin-dependent kinase inhibitors may play roles not only in regulating cell proliferation activity but also in regulating other functions such as differentiation in the lung during the foetal period.     

Cytokine-stimulated T lymphocyte proliferation is regulated by p27Kip1

T lymphocyte growth is regulated by the cyclin-dependent kinase inhibitor p27(Kip1). Mice deficient in p27(Kip1) have increased proliferative responses to multiple cytokines, including IL-2, IL-4, and IL-12, but not to anti-CD3. In the absence of p27(Kip1), T cells proliferate faster than control cells, as evidenced by increased [(3)H]thymidine uptake, increased cell growth and division, and an increased number of cells in S phase. Importantly, this regulation is specific for p27(Kip1) in T cells, because hyperproliferation of T cells from mice deficient in p21(Cip1/Waf1) was not observed. In vivo, there is an expansion of activated/memory CD4(+) cells in p27(Kip1)-deficient mice before and after immunization. Furthermore, Ag-stimulated spleen cells from immunized p27(Kip1)-deficient mice demonstrated increased proliferative responses to IL-2 and increased secretion of IFN-gamma. Although IL-4 stimulated proliferative responses are diminished in Stat6-deficient T cells, activated T cells from mice doubly deficient in both p27(Kip1) and Stat6 recover normal proliferative responses to IL-4. Together, these data firmly support a role for p27(Kip1) as a negative regulator of cytokine-stimulated T cell growth.     

Expression of cyclin-dependent kinase inhibitors in taste buds of mouse and hamster

Taste buds are specialized epithelial cell clusters in the oral squamous cell epithelium. Although taste buds have been reported to renew rapidly, the mechanism of cell cycle control in these specialized structures remains unresolved. To clarify the cell cycle status and role of cyclin-dependent kinase inhibitors (CDKI) for cell cycle control in the taste buds, we analyzed cell proliferation activity using bromodeoxyuridine (BrdU) and Ki-67 immunostainings and the expression of the Cip/Kip family of CDKI (p21Cip1, p27Kip1, and p57Kip2) in the circumvallate papillae of mouse and hamster. BrdU-positive cells were detected in the basal layer of the oral epithelium. In the taste buds, Ki-67-positive cells were seen in the basal area, with only a very few positive cells in the taste buds. Both p21Cip1 and p27Kip1 positive cells were seen in the suprabasal layer of the non-gustatory oral epithelium. In the taste buds, stronger p27Kip1 staining was detected than in the non-gustatory epithelium. Western blotting analysis revealed that p27Kip1 was abundant in the mucosal tissues from circumvallate papillae. Thus, our study suggests that the taste bud cells except for basal cells are post-mitotic cells and that the cell cycle arrest associated with taste bud cell differentiation could be regulated predominantly by p27Kip1.     

p57(Kip2) and p27(Kip1) cooperate to maintain hematopoietic stem cell quiescence through interactions with Hsc70

Cell cycle regulators play critical roles in the balance between hematopoietic stem cell (HSC) dormancy and proliferation. In this study, we report that cell cycle entry proceeded normally in HSCs null for cyclin-dependent kinase (CDK) inhibitor p57 due to compensatory upregulation of p27. HSCs null for both p57 and p27, however, were more proliferative and had reduced capacity to engraft in transplantation. We found that heat shock cognate protein 70 (Hsc70) interacts with both p57 and p27 and that the subcellular localization of Hsc70 was critical to maintain HSC cell cycle kinetics. Combined deficiency of p57 and p27 in HSCs resulted in nuclear import of an Hsc70/cyclin D1 complex, concomitant with Rb phosphorylation, and elicited severe defects in maintaining HSC quiescence. Taken together, these data suggest that regulation of cytoplasmic localization of Hsc70/cyclin D1 complex by p57 and p27 is a key intracellular mechanism in controlling HSC dormancy.     

Changes in p21(Cip1) and p27(Kip1) expression are not required for cell cycle entry and progression to S phase in Swiss 3T3 cells

The cyclin-dependent kinase inhibitors, p21(Cip1) and p27(Kip1), play an important role in the regulation of progression through G(1) to S phase in mammalian cells. Here we report that confluent 3T3 cells expressed p21(Cip1) and p27(Kip1) predominantly in the nucleus, and the level of both proteins declined as the cells entered the cell cycle and progressed through G(1) in response to serum growth factors. However, when confluent cells were serum starved prior to treatment, no downregulation of p21(Cip1) or p27(Kip1) expression was observed. Notably, serum starvation did not significantly influence the capacity of the cells to progress to the S phase. It was observed that serum starvation reduced cell density. Further, when cells were plated at a range of different densities, starved of serum to render them quiescent and then subsequently treated with serum, a reduction in p21(Cip1) and p27(Kip1) expression was observed in cells plated at high density but not in those at low density. Again, the extent and timing of progression to S phase was not influenced by cell density. To establish the potential role of cell:cell contact in the observed density-dependent regulation of p21(Cip1) and p27(Kip1) expression, cells were plated onto micorarrays of adhesive islands that prevented individual cells from making any contact with other cells. Under these conditions serum growth factors induced p21(Cip1) and p27(Kip1) downregulation, and hence, there is no requirement for cell:cell contact. Together, these data indicate that there are conditions under which 3T3 cells can progress to the S phase without downregulation of p21(Cip1) and p27(Kip1). The significance of these observations and mechanisms by which density-dependent regulation of p21(Cip1) and p27(Kip1) expression may occur are discussed.     

Mice lacking a CDK inhibitor, p57Kip2, exhibit skeletal abnormalities and growth retardation

p57Kip2, one of the cyclin-dependent kinase (CDK) inhibitors, has been suggested to be a tumor suppressor candidate. To elucidate its biological roles in mouse development and tumorigenesis, we created p57Kip2-deficient mice. The p57Kip2-deficient mice exhibited a cleft palate and defective bone formation resulting in severe dyspnea. Most of the p57Kip2-deficient mice died within 24 h after birth, while about 10% of them survived beyond the weaning period. All of the surviving mice showed severe growth retardation. The males showed immaturity of the testes, prostate and seminal vesicles, and the females showed vaginal atresia, immaturity of the uterus, and an increased number of atretic follicles. Although Yan et al. and Zhang et al. have already reported p57Kip2-deficient mice, they could not investigate the phenotypes of the surviving p57Kip2-deficient mice. Also, most of the symptoms of Beckwith-Wiedemann syndrome could not be reproduced in the mutant mice. Embryonic fibroblasts prepared from p57Kip2-deficient mice showed no differences in the proliferation rate and saturation density, suggesting that G1 arrest is largely independent of p57Kip2 function. Our results suggest that p57Kip2 plays a critical role in development, but do not support the hypothesis that the p57Kip2 gene is a tumor-suppressor gene or is responsible for Beckwith-Wiedemann syndrome.     

Phosphorylations of cyclin-dependent kinase 2 revisited using two-dimensional gel electrophoresis

To control the G1/S transition and the progression through the S phase, the activation of the cyclin-dependent kinase (CDK) 2 involves the binding of cyclin E then cyclin A, the activating Thr-160 phosphorylation within the T-loop by CDK-activating kinase (CAK), inhibitory phosphorylations within the ATP binding region at Tyr-15 and Thr-14, dephosphorylation of these sites by cdc25A, and release from Cip/Kip family (p27kip1 and p21cip1) CDK inhibitors. To re-assess the precise relationship between the different phosphorylations of CDK2, and the influence of cyclins and CDK inhibitors upon them, we introduce here the use of the high resolution power of two-dimensional gel electrophoresis, combined to Tyr-15- or Thr-160-phosphospecific antibodies. The relative proportions of the potentially active forms of CDK2 (phosphorylated at Thr-160 but not Tyr-15) and inactive forms (non-phosphorylated, phosphorylated only at Tyr-15, or at both Tyr-15 and Thr-160), and their respective association with cyclin E, cyclin A, p21, and p27, were demonstrated during the mitogenic stimulation of normal human fibroblasts. Novel observations modify the current model of the sequential CDK2 activation process: (i) Tyr-15 phosphorylation induced by serum was not restricted to cyclin-bound CDK2; (ii) Thr-160 phosphorylation engaged the entirety of Tyr-15-phosphorylated CDK2 associated not only with a cyclin but also with p27 and p21, suggesting that Cip/Kip proteins do not prevent CDK2 activity by impairing its phosphorylation by CAK; (iii) the potentially active CDK2 phosphorylated at Thr-160 but not Tyr-15 represented a tiny fraction of total CDK2 and a minor fraction of cyclin A-bound CDK2, underscoring the rate-limiting role of Tyr-15 dephosphorylation by cdc25A.