p57(Kip2) regulates progenitor cell proliferation and amacrine interneuron development in the mouse retina

A precise balance between proliferation and differentiation must be maintained during retinal development to obtain the correct proportion of each of the seven cell types found in the adult tissue. Cyclin kinase inhibitors can regulate cell cycle exit coincident with induction of differentiation programs during development. We have found that the p57(Kip2) cyclin kinase inhibitor is upregulated during G(1)/G(0) in a subset of retinal progenitor cells exiting the cell cycle between embryonic day 14.5 and 16.5 of mouse development. Retroviral mediated overexpression of p57(Kip2) in embryonic retinal progenitor cells led to premature cell cycle exit. Retinae from mice lacking p57(Kip2) exhibited inappropriate S-phase entry and apoptotic nuclei were found in the region where p57(Kip2) is normally expressed. Apoptosis precisely compensated for the inappropriate proliferation in the p57(Kip2)-deficient retinae to preserve the correct proportion of the major retinal cell types. Postnatally, p57(Kip2) was found to be expressed in a novel subpopulation of amacrine interneurons. At this stage, p57(Kip2 )did not regulate proliferation. However, perhaps reflecting its role during this late stage of development, animals lacking p57(Kip2) showed an alteration in amacrine subpopulations. p57(Kip2) is the first gene to be implicated as a regulator of amacrine subtype/subpopulation development. Consequently, we propose that p57(Kip2) has two roles during retinal development, acting first as a cyclin kinase inhibitor in mitotic progenitor cells, and then playing a distinct role in neuronal differentiation.     

p57(Kip2) and cancer: time for a critical appraisal

p57(Kip2) is a cyclin-dependent kinase inhibitor belonging to the Cip/Kip family, which also includes p21(Cip1) and p27(Kip1). So far, p57(Kip2) is the least-studied Cip/Kip protein, and for a long time its relevance has been related mainly to its unique role in embryogenesis. Moreover, genetic and molecular studies on animal models and patients with Beckwith-Wiedemann syndrome have shown that alterations in CDKN1C (the p57(Kip2) encoding gene) have functional relevance in the pathogenesis of this disease. Recently, a number of investigations have identified and characterized heretofore unexpected roles for p57(Kip2). The protein appears to be critically involved in initial steps of cell and tissue differentiation, and particularly in neuronal development and erythropoiesis. Intriguingly, p27(Kip1), the Cip/Kip member that is most homologous to p57(Kip2), is primarily involved in the process of cell cycle exit. p57(Kip2) also plays a critical role in controlling cytoskeletal organization and cell migration through its interaction with LIMK-1. Furthermore, p57(Kip2) appears to modulate genome expression. Finally, accumulating evidence indicates that p57(Kip2) protein is frequently downregulated in different types of human epithelial and nonepithelial cancers as a consequence of genetic and epigenetic events. In summary, the emerging picture is that several aspects of p57(Kip2)'s functions are only poorly clarified. This review represents an appraisal of the data available on the p57(Kip2) gene and protein structure, and its role in human physiology and pathology. We particularly focus our attention on p57(Kip2) changes in cancers and pharmacological approaches for modulating p57(Kip2) levels.     

Regulation of the Cyclin-Dependent Kinase Inhibitor p57Kip2 Expression by p63

The cyclin-dependent kinase (CDK) inhibitor p57^Kip2 is a negative regulator of cell proliferation, binding to a variety of cyclin-CDK complexes and inhibiting their kinase activities. The p57^Kip2 gene was recognized as a target gene for p73β, one member of the p53 family. In spite of this, the phenotypes of p73 and p57Kip2 knock out mice do not resemble each other while there is a phenotypic overlap betweeen the p57^Kip2 null mice, the p63 null mice and patients affected by p63 associated syndromes, suggesting that p57^Kip2 could be indeed a downstream target of p63. By ChIP we determined that in the HaCaT cell line the δNp63α protein is associated to three different regions of the p57Kip2 gene. δNp63 can activate both the endogenous p57^Kip2 gene and a reporter vector containing a -2191 promoter fragment of the p57^Kip2 gene. Natural p63 mutants, associated to the AEC syndrome, show a partial or complete lack of transactivation potential of the p57^Kip2 promoter, while three other natural p63 mutants, associated to the EEC, LMS and SHFM-4 syndromes, were less affected. These data suggests that p63 play an important role in the regulation of p57^Kip2 expression and that this regulation is subverted in AEC p63 mutants.     

Deregulation of the p57-E2F1-p53 axis results in nonobstructive hydrocephalus and cerebellar malformation in mice

The cyclin-dependent kinase inhibitor (CKI) p57(Kip2) plays a pivotal role in cell cycle arrest during development, in particular, in the regulation of the entry of proliferating progenitors into quiescence. The gene encoding p57 undergoes genomic imprinting, and impairment of the regulation of p57 expression results in various developmental anomalies in humans and mice. We now show that p57 is expressed predominantly in the subcommissural organ and cerebellar interneurons in the mouse brain and that mice with brain-specific deletion of the p57 gene (Kip2) manifest prominent nonobstructive hydrocephalus as well as cerebellar malformation associated with the loss of Pax2-positive interneuron precursors and their descendants, including Golgi cells and γ-aminobutyric acid-containing neurons of the deep cerebellar nuclei. These abnormalities were found to be attributable to massive apoptosis of precursor cells in the developing brain. The morphological defects of the p57-deficient mice were corrected by knock-in of the gene for the related CKI p27(Kip1) at the Kip2 locus. The abnormalities were also prevented by additional genetic ablation of p53 or E2F1. Our results thus implicate p57 in cell cycle arrest in the subcommissural organ and Pax2-positive interneuron precursors, with the lack of p57 resulting in induction of p53-dependent apoptosis due to hyperactivation of E2F1.     

p57 is required for quiescence and maintenance of adult hematopoietic stem cells

Quiescence is required for the maintenance of hematopoietic stem cells (HSCs). Members of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors (p21, p27, p57) have been implicated in HSC quiescence, but loss of p21 or p27 in mice affects HSC quiescence or functionality only under conditions of stress. Although p57 is the most abundant family member in quiescent HSCs, its role has remained uncharacterized. Here we show a severe defect in the self-renewal capacity of p57-deficient HSCs and a reduction of the proportion of the cells in G(0) phase. Additional ablation of p21 in a p57-null background resulted in a further decrease in the colony-forming activity of HSCs. Moreover, the HSC abnormalities of p57-deficient mice were corrected by knocking in the p27 gene at the p57 locus. Our results therefore suggest that, among Cip/Kip family CDK inhibitors, p57 plays a predominant role in the quiescence and maintenance of adult HSCs.     

p57KIP2 modulates stress-activated signaling by inhibiting c-Jun NH2-terminal kinase/stress-activated protein Kinase

p57KIP2, a member of the Cip/Kip family of enzymes that inhibit several cyclin-dependent kinases, plays a role in many biological events including cell proliferation, differentiation, apoptosis, tumorigenesis and developmental changes. The human p57KIP2 gene is located in chromosome 11p15.5, a region implicated in sporadic cancers and Beckwith-Wiedemann syndrome. We here report that p57KIP2 physically interacts with and inhibits c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK). The carboxyl-terminal QT domain of p57KIP2 is crucial for the inhibition of JNK/SAPK. Overexpressed p57KIP2 also suppressed UV- and MEKK1-induced apoptotic cell death. p57KIP2 expression during C2C12 myoblast differentiation resulted in repression of the JNK activity stimulated by UV light. Furthermore, UV-stimulated JNK1 activity was higher in mouse embryonic fibroblasts derived from p57-/- mice than in the cells from wild-type mice. Taken together, these findings suggest that p57KIP2 modulates stress-activated signaling by functioning as an endogenous inhibitor of JNK/SAPK.     

P57KIP2 targeted disruption and beckwith-wiedemann syndrome: Is the inhibitor just a contributor?

Beckwith-Wiedemann syndrome is a human congenital disorder characterized by a wide variety of growth abnormalities, including developmental defects and predisposition to certain tumors. Genetic evidence has suggested a role for p57^KIP2, a member of a family of cell cycle inhibitory genes, in Beckwith-Wiedemann syndrome. Two independent groups^(1,2) have reported the generation and characterization of mice lacking functional p57^KIP2, These mice demonstrate a number of abnormal phenotypes which overlap with, although do not completely recapitulate, Beckwith-Wiedemann syndrome. These findings advance the molecular characterization of a human disorder, and provide insight into the interplay between regulation of cell division and development.     

Multiple Mechanisms Regulate Imprinting of the Mouse Distal Chromosome 7 Gene Cluster

Genomic imprinting is an epigenetic process that results in the preferential silencing of one of the two parental copies of a gene. Although the precise mechanisms by which genomic imprinting occurs are unknown, the tendency of imprinted genes to exist in chromosomal clusters suggests long-range regulation through shared regulatory elements. We characterize a 800-kb region on the distal end of mouse chromosome 7 that contains a cluster of four maternally expressed genes, H19, Mash2, Kvlqt1, and p57^Kip2, as well as two paternally expressed genes, Igf2 and Ins2, and assess the expression and imprinting of Mash2, Kvlqt1, and p57^Kip2 during development in embryonic and extraembryonic tissues. Unlike Igf2 and Ins2, which depend on H19 for their imprinting, Mash2, p57^Kip2, and Kvlqt1 are unaffected by a deletion of the H19 gene region, suggesting that these more telomeric genes are not regulated by the mechanism that controls H19, Igf2, and Ins2. Mutations in human p57^Kip2 have been implicated in Beckwith-Wiedemann syndrome, a disease that has also been associated with loss of imprinting of IGF2. We find, however, that a deletion of the gene has no effect on imprinting within the cluster. Surprisingly, the three maternally expressed genes are regulated very differently by DNA methylation; p57^Kip2 is activated, Kvlqt1 is silenced, and Mash2 is unaffected in mice lacking DNA methyltransferase. We conclude that H19 is not a global regulator of imprinting on distal chromosome 7 and that the telomeric genes are imprinted by a separate mechanism(s).     

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.     

The homeostasis but not the differentiation of T cells is regulated by p27(Kip1)

The cyclin-dependent kinase inhibitor p27(Kip1) is a critical regulator of T cell proliferation. To further examine the relationship of T cell proliferation and differentiation, we examined the ability of T cells deficient in p27(Kip1) to differentiate into Th subsets. We observed increased Th2 differentiation in p27(Kip1)-deficient cultures. In addition to increases in CD4(+) and CD8(+) T cells, there is a similar increase in gamma delta T cells in p27(Kip1)-deficient mice compared with wild-type mice. The increase in Th2 differentiation is correlated to an increase of IL-4 secretion by CD4(+)DX5(+)TCR alpha beta(+)CD62L(low) T cells but not to increased expansion of differentiating Th2 cells. While STAT4- and STAT6-deficient T cells have diminished proliferative responses to IL-12 and IL-4, respectively, proliferative responses are increased in T cells doubly deficient in p27(Kip1) and STAT4 or STAT6. In contrast, the increased proliferation and differentiative capacity of p27(Kip1)-deficient T cells has no effect on the ability of STAT4/p27(Kip1)- or STAT6/p27(Kip1)-deficient CD4(+) cells to differentiate into Th1 or Th2 cells, respectively. Thus, while p27(Kip1) regulates the expansion and homeostasis of several T cell subsets, it does not affect the differentiation of Th subsets.     

Increased expression of vascular endothelial growth factor in placentas of p57(Kip2) null embryos

Placentas of mice lacking p57(Kip2) expression have trophoblastic hyperplasia. To elucidate the mechanism underlying this phenomenon, we studied expression of two angiogenic factors, vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). Immunohistochemical analysis with anti-VEGF antibodies indicated that VEGF expression was stronger and more clearly detectable in placentas of p57(Kip2) null embryos compared to wild-type placentas. PlGF showed no significant differences between placentas of p57(Kip2) null and wild-type embryos. In quantitative analysis, placentas of p57(Kip2) null embryos showed higher VEGF messenger (m)RNA and protein levels than did wild-type placentas. PlGF mRNA and protein levels were not significantly different. These findings suggest that VEGF is involved in the hyperplasia that occurs in placentas of p57(Kip2) null embryos.     

Microdeletion of LIT1 in familial Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS), which causes prenatal overgrowth, midline abdominal wall defects, macroglossia, and embryonal tumors, is a model for understanding the relationship between genomic imprinting, human development, and cancer. The causes are heterogeneous, involving multiple genes on 11p15 and including infrequent mutation of p57(KIP2) or loss of imprinting of either of two imprinted gene domains on 11p15: LIT1, which is near p57(KIP2), or H19/IGF2. Unlike Prader-Willi and Angelman syndromes, no chromosomal deletions have yet been identified. Here we report a microdeletion including the entire LIT1 gene, providing genetic confirmation of the importance of this gene region in BWS. When inherited maternally, the deletion causes BWS with silencing of p57(KIP2), indicating deletion of an element important for the regulation of p57(KIP2) expression. When inherited paternally, there is no phenotype, suggesting that the LIT1 RNA itself is not necessary for normal development in humans.     

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.     

The cell cycle inhibitor p57(Kip2) promotes cell death via the mitochondrial apoptotic pathway

The p57(Kip2) gene belongs to the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors and has been suggested to be a tumor suppressor gene, being inactivated in various types of human cancers. However, little is known concerning p57(Kip2) possible interplay with the apoptotic cell death machinery and its possible implication for cancer. Here, we report that selective p57(Kip2) expression sensitizes cancer cells to apoptotic agents such as cisplatin, etoposide and staurosporine (STS) via a mechanism, which does not require p57(Kip2)-mediated inhibition of CDK. Translocation of p57(Kip2) to mitochondria occurs within 20 min after STS application. In fact, p57(Kip2) primarily promotes the intrinsic apoptotic pathways, favoring Bax activation and loss of mitochondrial transmembrane potential, consequent release of cytochrome-c into cytosol, caspase-9 and caspase-3 activation. In accordance, Bcl2 overexpression or voltage-dependent anion channel (VDAC) inhibition is able to inhibit p57(Kip2) cell death promoting effect. Thus, in addition to its established function in control of proliferation, these results reveal a mechanism whereby p57(Kip2) influences the mitochondrial apoptotic cell death pathway in cancer cells.     

The Cdk inhibitor p21 is required for necrosis,but it inhibits apoptosis following toxin-induced liver injury

Liver injury and repair were examined in wild type, p21Waf1/Cip1, and p27Kip1-deficient mice following carbon tetrachloride (CCl4) administration. In wild type liver, p21 expression is induced in a biphasic manner following injection of CCl4, with an early peak of p21 expression occurring in pericentral hepatocytes at 6 h, prior to evidence of injury, and a second peak succeeding regenerative proliferation. In contrast, p27 is present throughout the quiescent liver, but its expression decreases following CCl4 injection. Surprisingly, p21-deficient animals were resistant to CCl4-induced necrotic injury, indicating that rapid induction of p21 in pericentral hepatocytes following CCl4 injection contributes to subsequent necrosis. Expression of cytochrome P450 2E1, which plays an essential role in CCl4-induced necrotic injury, was not affected in p21-deficient mice. Although they had the least injury, p21-deficient mice had the highest levels of hepatic proliferation that correlated with increases in hyperphosphorylated retinoblastoma protein and Cyclin A gene expression. Increased replication in p21-deficient livers was counteracted by an increase in hepatocyte apoptosis as detected by caspase-3 activation. p21 plays distinct and opposing roles regulating hepatocyte survival during injury and subsequent repair, with early induction of p21 contributing to necrotic injury and later expression to cessation of proliferation and hepatocyte survival.     

Signal-induced ubiquitination of p57(Kip2) is independent of the C-terminal consensus Cdk phosphorylation site

The cyclin-dependent kinase inhibitor p57(Kip2) is required for normal mouse embryonic development. p57(Kip2) consists of four structurally distinct domains in which the conserved C-terminal nuclear targeting domain contains a putative Cdk phosphorylation site (Thr(342)) that shares a great similitude in the adjacent sequences with p27(Kip1) but not with p21(Cip1). Phosphorylation on Thr(187) has been shown to promote degradation of p27(Kip1). Although there is sequence homology between the C-terminal part of p27(Kip1) and p57(Kip2), we show that the ubiquitination and degradation of p57(Kip2) are independent of Thr(342). In contrast a destabilizing element located in the N-terminal is implicated in p57(Kip2) destabilization.