Receptor-Ck regulates the expression of cyclin-dependent kinase inhibitors (p16; p27)

The present study was addressed to understand the interrelationship between Receptor-C_k induction-activation coupling; isoprenoids (derived from mevalonate pathway) and cyclin-dependent kinase inhibitors. The results reported here unambiguously reveal that isoprenoids regulate the expression of genes coding for CDK inhibited p16 and p27. Further, Receptor-C_k dependent signalling, known to control the mevalonate pathway, had a direct effect upon the p27 gene expression. Based upon these studies coupled with our earlier findings we propose that Receptor-C_k has an important role in the regulation of cell cycle machinery.     

The cyclin-dependent kinase inhibitors p57 and p27 regulate neuronal migration in the developing mouse neocortex

Neuronal precursors remain in the proliferative zone of the developing mammalian neocortex until after they have undergone neuronal differentiation and cell cycle arrest. The newborn neurons then migrate away from the proliferative zone and enter the cortical plate. The molecules that coordinate migration with neuronal differentiation have been unclear. We have proposed in this study that the cdk inhibitors p57 and p27 play a role in this coordination. We have found that p57 and p27 mRNA increase upon neuronal differentiation of neocortical neuroepithelial cells. Knockdown of p57 by RNA interference resulted in a significant delay in the migration of neurons that entered the cortical plate but did not affect neuronal differentiation. Knockdown of p27 also inhibits neuronal migration in the intermediate zone as well as in the cortical plate, as reported by others. We have also found that knockdown of p27 increases p57 mRNA levels. These results suggest that both p57 and p27 play essential roles in neuronal migration and may, in concert, coordinate the timing of neuronal differentiation, migration, and possibly cell cycle arrest in neocortical development.     

Deficiency of cyclin-dependent kinase inhibitors p21 and p27 accelerates atherogenesis in apolipoprotein E-deficient mice

Cyclin-dependent kinase inhibitors, p21^Cip1 and p27^Kip1, are upregulated during vascular cell proliferation and negatively regulate growth of vascular cells. We hypothesized that absence of either p21^Cip1 or p27^Kip1 in apolipoprotein E (apoE)-deficiency may increase atherosclerotic plaque formation. Compared to apoE^−/− aortae, both apoE^−/−/p21^−/− and apoE^−/−/p27^−/− aortae exhibited significantly more atherosclerotic plaque following a high-cholesterol regimen. This increase was particularly observed in the abdominal aortic regions. Deficiency of p27^Kip1 accelerated plaque formation significantly more than p21^−/− in apoE^−/− mice. This increased plaque formation was in parallel with increased intima/media area ratios. Deficiency of p21^Cip1 and p27^Kip1 accelerates atherogenesis in apoE^−/− mice. These findings have significant implications for our understanding of the molecular basis of atherosclerosis associated with excessive proliferation of vascular cells.     

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.     

Bacterial cyclomodulin Cif blocks the host cell cycle by stabilizing the cyclin-dependent kinase inhibitors p21 and p27

The cycle inhibiting factor (Cif) is a cyclomodulin produced by enteropathogenic and enterohemorrhagic Escherichia coli. Upon injection into the host cell by the bacterial type III secretion system, Cif inhibits the G2/M transition via sustained inhibition of the mitosis inducer CDK1 independently of the DNA damage response. In this study, we show that Cif induces not only G2, but also G1 cell cycle arrest depending on the stage of cells in the cell cycle during the infection. In various cell lines including differentiated and untransformed enterocytes, the cell cycle arrests are correlated with the accumulation of the cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1). Cif-induced cyclin-dependent kinase inhibitor accumulation is independent of the p53 pathway but occurs through inhibition of their proteasome-mediated degradation. Our results provide a direct link between the mode of action of Cif and the host cell cycle control.     

Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts

The irreversible G1 arrest in senescent human diploid fibroblasts is probably caused by inactivation of the G1 cyclin-cyclin-dependent kinase (Cdk) complexes responsible for phosphorylation of the retinoblastoma protein (pRb). We show that the Cdk inhibitor p21(Sdi1,Cip1,Waf1), which accumulates progressively in aging cells, binds to and inactivates all cyclin E-Cdk2 complexes in senescent cells, whereas in young cells only p21-free Cdk2 complexes are active. Furthermore, the senescent-cell-cycle arrest occurs prior to the accumulation of the Cdk4-Cdk6 inhibitor p16(Ink4a), suggesting that p21 may be sufficient for this event. Accordingly, cyclin D1-associated phosphorylation of pRb at Ser-780 is lacking even in newly senescent fibroblasts that have a low amount of p16. Instead, the cyclin D1-Cdk4 and cyclin D1-Cdk6 complexes in these cells are associated with an increased amount of p21, suggesting that p21 may be responsible for inactivation of both cyclin E- and cyclin D1-associated kinase activity at the early stage of senescence. Moreover, even in the late stage of senescence when p16 is high, cyclin D1-Cdk4 complexes are persistent, albeit reduced by 相似文献   

The expression of p18INK4 and p27kip1 cyclin-dependent kinase inhibitors is regulated differently during human B cell differentiation

Cell cycle progression is under the control of cyclin-dependent kinases (cdks), the activity of which is dependent on the expression of specific cdk inhibitors. In this paper we report that the two cdk inhibitors, p27(Kip1) and p18(INK4c), are differently expressed and control different steps of human B lymphocyte activation. Resting B cells contain large amounts of p27(Kip1) and no p18(INK4c). In vitro stimulation by Staphylococcus aureus Cowan 1 strain or CD40 ligand associated with IL-10 and IL-2 induces a rapid decrease in p27(Kip1) expression combined with cell cycle entry and progression. In contrast, in vitro Ig production correlates with specific expression of p18(INK4c) and early G(1) arrest. This G(1) arrest is associated with inhibition of cyclin D3/cdk6-mediated retinoblastoma protein phosphorylation by p18(INK4c). A similar contrasting pattern of p18(INK4c) and p27(Kip1) expression is observed both in B cells activated in vivo and in various leukemic cells. Expression of p18(INK4c) was also detected in various Ig-secreting cell lines in which both maximum Ig secretion and specific p18(INK4c) expression were observed during the G(1) phase. Our study shows that p27(Kip1) and p18(INK4c) have different roles in B cell activation; p27(Kip1) is involved in the control of cell cycle entry, and p18(INK4c) is involved in the subsequent early G(1) arrest necessary for terminal B lymphocyte differentiation.     

A naturally occurring mixture of tocotrienols inhibits the growth of human prostate tumor,associated with epigenetic modifications of cyclin-dependent kinase inhibitors p21 and p27

Tocotrienols, members of the vitamin E family, have three unsaturated bonds in their side chains. Recently, it has been suggested that the biological effects of tocotrienols may differ from that of tocopherols. Several in vitro studies have shown that tocotrienols have stronger anticancer effects than tocopherols. VCaP cell line used in this study is from a vertebral bone metastasis from a patient with prostate cancer. Eight-week-old male NCr(−/−) nude mice were subcutaneously injected with VCaP-luc cells in matrigel and then administered a tocotrienol mixture for 8 weeks. The tocotrienol mixture inhibited the growth of human prostate tumor xenografts in a dose-dependent manner. The concentrations of tocotrienols and their metabolites were significantly increased in treatment groups. Tocotrienols inhibited prostate tumor growth by suppressing cell proliferation, which was associated with the induction of the cyclin-dependent kinase (CDK) inhibitors p21 and p27. In addition, tocotrienol treatment was associated with elevated H3K9 acetylation levels at proximal promoter regions of p21 and p27 and with decreased expression of histone deacetylases. Tocotrienols inhibited human prostate tumor growth, associated with up-regulation of the CDK inhibitors p21 and p27. Elevated expression of p21 and p27 could be partly due to the suppressed expression of HDACs.     

Identification of Xenopus cyclin-dependent kinase inhibitors, p16Xic2 and p17Xic3

The Cip/Kip family of mammalian cyclin-dependent kinase (cdk) inhibitors plays important roles in development, particularly in cell fate determination and differentiation, in addition to their function of blocking cell cycle progression. We have identified two novel members of the Kip/Cip cdk inhibitor family, p16Xic2 and p17Xic3, from Xenopus laevis. Sequence analysis revealed that p16Xic2 and p17Xic3 are orthologues of mammalian p21Cip1 and p27Kip1, respectively. Overexpression of these inhibitors results in cell cycle arrest by inhibition of cdk2 activity. Interestingly, the expression of these inhibitors is highly developmentally regulated. p16Xic2 is highly expressed in differentiating somite, tail bud, lens, and cement gland, while p17Xic3 is expressed in the central nervous system. In a retinal cell fate determination assay, both p16Xic2 and p17Xic3 have an activity that influences cell fate determination. These observations suggest that p16Xic2 and p17Xic3 might be involved in cell fate determination in a tissue-specific manner by coordinating proliferation and differentiation as observed with p27Xic1.     

p27kip1 (cyclin-dependent kinase inhibitor 1B) controls ovarian development by suppressing follicle endowment and activation and promoting follicle atresia in mice

In humans, the molecular mechanisms underlying ovarian follicle endowment and activation, which are closely related to the control of female reproduction, occurrence of menopause, and related diseases such as premature ovarian failure, are poorly understood. In the current study, we provide several lines of genetic evidence that the cyclin-dependent kinase (Cdk) inhibitor 1B (commonly known as p27(kip1) or p27) controls ovarian development in mice by suppressing follicle endowment and activation, and by promoting follicle death. In p27-deficient (p27(-/-)) mice, postnatal follicle assembly was accelerated, and the number of endowed follicles was doubled as compared with p27(+/+) mice. Moreover, in p27(-/-) ovaries the primordial follicle pool was prematurely activated once it was endowed, and at the same time the massive follicular death that occurs before sexual maturity was rescued by loss of p27. In early adulthood, however, the overactivated follicular pool in p27(-/-) ovaries was largely depleted, causing premature ovarian failure. Furthermore, we have extensively studied the molecular mechanisms underlying the above-mentioned phenotypes seen in p27(-/-) ovaries and have found that p27 controls follicular development by several distinct mechanisms at different stages of development of the ovary. For example, p27 controls oocyte growth by suppressing the functions of Cdk2/Cdc2-cyclin A/E1 in oocytes that are arrested at the diplotene stage of meiosis I. This function of p27 is distinct from its well-known role as a suppressor of cell cycle progression. In addition, we have found that p27 activates the caspase-9-caspase-3-caspase-7-poly (ADP-ribose) polymeraseapoptotic cascade by inhibiting Cdk2/Cdc2-cyclin A/B1 kinase activities in follicles, thereby inducing follicle atresia. Our results suggest that the p27 gene is important in determining mammalian ovarian development. This study therefore provides insight into ovary-borne genetic aberrations that cause defects in folliculogenesis and infertility in humans.     

The cyclin-dependent kinase inhibitors p15INK4B and p21CIP1 are critical regulators of fibrillar collagen-induced tumor cell cycle arrest

The extracellular matrix is a crucial component in determining cell fate. Fibrillar collagen in its native form inhibits cell proliferation, whereas in its monomeric form it stimulates proliferation. The observation of elevated levels of p27(KIP1) in cells plated in the presence of fibrillar collagen has led to the assumption that this kinase inhibitor was responsible for cell cycle arrest on fibrillar collagen. Here we provide evidence that p15(INK4b), rather than p27(KIP1), is the cyclin-dependent kinase inhibitor responsible for G0/G1 arrest of human melanoma cells grown on fibrillar collagen. Additionally, we demonstrate that fibrillar collagen can also arrest cells at the G2 phase, which is mediated in part by p21(CIP1). Our data, in addition to identifying cyclin-dependent kinase inhibitors important in cell cycle arrest mediated by fibrillar collagen, demonstrate the complexity of cell cycle regulation and indicate that modulating a single cyclin-dependent kinase inhibitor does not disrupt cell proliferation in the presence of fibrillar collagen.     

Induction of anergy in Th1 cells associated with increased levels of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1

Th1 cells exposed to Ag and the G(1) blocker n-butyrate in primary cultures lose their ability to proliferate in Ag-stimulated secondary cultures. The ability of n-butyrate to induce anergy in Ag-stimulated, but not resting, Th1 cells was shown here to be blocked by cycloheximide. Subsequent experiments to delineate the nature of the protein apparently required for n-butyrate-induced Th1 cell anergy focused on the role of cyclin-dependent kinase (cdk) inhibitors p21(Cip1) and p27(Kip1). Normally, entry into S phase by Th1 cells occurs around 24 h after Ag stimulation and corresponds with relatively low levels of both p21(Cip1) and p27(Kip1). However, unlike control Th1 cells, anergic Th1 cells contained high levels of both p21(Cip1) and p27(Kip1) when examined 24 h after Ag stimulation. The increase in p21(Cip1) observed in Ag-stimulated anergic Th1 cells appeared to be initiated in primary cultures. In contrast, the increase in p27(Kip1) observed in these anergic Th1 cells appears to represent a re-expression of the protein much earlier than control cells following Ag stimulation in secondary cultures. The anergic Th1 cells contained functionally active cdk inhibitors capable of inhibiting the activity of both endogenous and exogenous cdks. Consequently, it appears that n-butyrate-induced anergy in Th1 cells correlated with the up-regulation of p21(Cip1) and perhaps the downstream failure to maintain low levels of p27(Kip1). Increased levels of both p21(Cip1) and p27(Kip1) at the end of G(1) could prevent cdk-mediated entry into S phase, and thus help maintain the proliferative unresponsiveness found in the anergic Th1 cells.     

The tuberous sclerosis genes and regulation of the cyclin-dependent kinase inhibitor p27

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor syndrome that affects approximately 1 in 6000 individuals. It is characterized by the development of tumors, named hamartomas, in the kidneys, heart, skin and brain. The latter often cause seizures, mental retardation, and a variety of developmental disorders, including autism. This disease is caused by mutations within the tumor suppressor gene TSC1 on chromosome 9q34 encoding hamartin or within TSC2 on chromosome 16p13.3 encoding tuberin. TSC patients carry a mutant TSC1 or TSC2 gene in each of their somatic cells, and loss of heterozygosity has been documented in a wide variety of TSC tumors. Recent data suggest that functional inactivation of TSC proteins might also be involved in the development of other diseases not associated with TSC, such as sporadic bladder cancer, breast cancer, ovarian carcinoma, gall bladder carcinoma, non-small-cell carcinoma of the lung, and Alzheimer's disease. Tuberin and hamartin form a heterodimer, suggesting they might affect the same processes. Tuberin is assumed to be the functional component of the complex and has been implicated in the regulation of different cellular functions. The TSC proteins regulate cell size control due to their involvement in the insulin signalling pathway. Furthermore, they are potent positive regulators of the cyclin-dependent kinase inhibitor p27, a major regulator of the mammalian cell cycle. Here we review the current knowledge on how mutations within the TSC genes could trigger deregulation of stability and localization of the tumor suppressor p27.     

Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27.

The p27(Kip1) protein associates with G1-specific cyclin-CDK complexes and inhibits their catalytic activity. p27(Kip1) is regulated at various levels, including translation, degradation by the ubiquitin/proteasome pathway and non-covalent sequestration. Here, we describe point mutants of p27 deficient in their interaction with either cyclins (p27(c-)), CDKs (p27(k-)) or both (p27(ck-)), and demonstrate that each contact is critical for kinase inhibition and induction of G1 arrest. Through its intact cyclin contact, p27(k-) associated with active cyclin E-CDK2 and, unlike wild type p27, p27(c-) or p27(ck-), was efficiently phosphorylated by CDK2 on a conserved C-terminal CDK target site (TPKK). Retrovirally expressed p27(k-) was rapidly degraded through the proteasome in Rat1 cells, but was stabilized by secondary mutation of the TPKK site to VPKK. In this experimental setting, exogenous wild-type p27 formed inactive ternary complexes with cellular cyclin E-CDK2, was not degraded through the proteasome, and was not further stabilized by the VPKK mutation. p27(ck-), which was not recruited to cyclin E-CDK2, also remained stable in vivo. Thus, selective degradation of p27(k-) depended upon association with active cyclin E-CDK2 and subsequent phosphorylation. Altogether, these data show that p27 must be phosphorylated by CDK2 on the TPKK site in order to be degraded by the proteasome. We propose that cellular p27 must also exist transiently in a cyclin-bound non-inhibitory conformation in vivo.     

Purification and crystallization of cyclin-dependent kinase inhibitor p21.

p21, a universal inhibitor of mammalian cyclin-dependent kinases (CDK), regulates cell cycle progression by forming various distinct protein complexes with cyclins, CDKs, and the proliferating cell nuclear antigen. We have overexpressed recombinant human p21 in E. coli and purified active p21 to near homogeneity on a large scale. Crystals of recombinant p21 have been grown in the space group P2(1) a = 157.4, b = 152.7, c = 90.6 A, and beta = 92.7 degrees. The diffraction data of the recombinant p21 have been collected to 2.5 and 3.5 A resolution for the native crystal and two heavy atom derivatives of mercury and iridium.     

The role of p21-activated kinase in the initiation of atherosclerosis

ABSTRACT: BACKGROUND: p21-activated kinase (PAK) has been implicated in the inflammatory activation of endothelial cells by disturbed fluid shear stress, which is the initiating stimulus in atherosclerosis. The study addresses whether PAK1 contributes to inflammatory marker expression in endothelial cells at atherosclerosis-susceptible regions of arteries in vivo. METHOD: Aortas from WT and PAK1-/- C57BL/6J mice on a normal chow diet were fixed, dissected and processed for immunohistochemistry using a panel of inflammatory markers. We visualized and quantified staining in the endothelium at the greater and lesser curvatures of the arch of aorta, as atherosclerosis-resistant and susceptible regions, respectively. RESULTS: Fibronectin, VCAM-1 and the activated RelA NF-kappaB subunit were localized to the lesser curvature and decreased in PAK1-/- mice. The activated RelB NF-kappaB subunit was also localized to the lesser curvature but was increased in PAK1-/- mice. Low levels of staining for ICAM-1 and the monocyte/macrophage marker Mac2 indicated that overall inflammation in this tissue was minimal. CONCLUSION: These data show that PAK1 has a significant pro-inflammatory function at atherosclerosisprone sites in vivo. These effects are seen in young mice with very low levels of inflammation, suggesting that inflammatory activation of the endothelium is primarily biomechanical. Activation involves NF-kappaB, expression of leukocyte recruitment receptors and fibronectin deposition. These results support and extend in vitro studies demonstrating that PAK contributes to activation of inflammatory pathways in endothelial cells by fluid shear stress.