The human ubiquitin-conjugating enzyme Cdc34 controls cellular proliferation through regulation of p27Kip1 protein levels

Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27Kip1 was shown to be required for the activation of key cyclin-dependent kinases, thereby triggering the onset of DNA replication and cell cycle progression. Although the SCFSkp2 ubiquitin ligase has been reported to mediate p27Kip1 degradation, the nature of the human ubiquitin-conjugating enzyme involved in this process has not yet been determined at the cellular level. Here, we show that antisense oligonucleotides targeting the human ubiquitin-conjugating enzyme Cdc34 downregulate its expression, inhibit the degradation of p27Kip1, and prevent cellular proliferation. Elevation of p27Kip1 protein level is found to be the sole requirement for the inhibition of cellular proliferation induced upon downregulation of Cdc34. Indeed, reducing the expression of p27Kip1 with a specific antisense oligonucleotide is sufficient to reverse the anti-proliferative phenotype elicited by the Cdc34 antisense. Furthermore, downregulation of Cdc34 is found to specifically increase the abundance of the SCFSkp2) ubiquitin ligase substrate p27Kip1, but has no concomitant effect on the level of IkBalpha and beta-catenin, which are known substrates of a closely related SCF ligase.     

Increased p27Kip1 cyclin-dependent kinase inhibitor gene expression following anti-IgM treatment promotes apoptosis of WEHI 231 B cells

Engagement of the B cell receptor of WEHI 231 immature B cells leads sequentially to a drop in c-Myc, to induction of the cyclin-dependent kinase inhibitor p27Kip1, and finally to apoptosis. Recently we demonstrated that the drop in c-Myc expression promotes cell death, whereas the induction of p27 has been shown to lead to growth arrest. In this paper, we demonstrate that increased p27 expression also promotes apoptosis of WEHI 231 B cells. The rescue of WEHI 231 cells by CD40 ligand engagement of its receptor prevented the increase in p27 induction. Inhibition of p27-ablated apoptosis induced upon expression of antisense c-myc RNA. Furthermore, specific induction of p27 gene expression resulted in apoptosis of WEHI 231 cells. Lastly, inhibition of expression of c-Myc, upon induction of an antisense c-myc RNA vector, was sufficient to induce increased p27 levels and apoptosis. Thus, these findings define a signaling pathway during B cell receptor engagement in which the drop in c-Myc levels leads to an increase in p27 levels that promotes apoptosis.     

Hepatitis B virus pre-S2 mutant surface antigen induces degradation of cyclin-dependent kinase inhibitor p27Kip1 through c-Jun activation domain-binding protein 1

The hepatitis B virus (HBV) large surface antigen (LHBS) mutant with deletion at the pre-S(2) region accumulates in endoplasmic reticulum (ER) and is associated with HBV-induced hepatocellular carcinogenesis. In this study, we found that the pre-S(2) LHBS mutant directly interacts with the Jun activation domain-binding protein 1 (JAB1). Association of pre-S(2) LHBS with JAB1 dissociated JAB1 from the JAB1/IRE1 complex in ER. The free (active) JAB1 then translocated into cell nuclei and rendered the Cdk inhibitor p27(Kip1) to cytosolic proteasome for degradation. The pre-S(2) LHBS mutant induced hyperphosphorylation of tumor suppressor retinoblastoma (RB) via cyclin-dependent kinase 2 (Cdk2), a downstream molecule regulated by p27(Kip1). This effect is independent of the ER stress signaling pathway. The transgenic mice carrying the pre-S(2) mutant LHBS gene also exhibited Cdk2 activation, p27(Kip1) degradation, as well as RB hyperphosphorylation. The mouse hepatocytes exhibited morphologic abnormalities such as chromatin condensation, multinucleation, and dysplasia of hepatocytes. In summary, the pre-S(2) LHBS mutant causes p27(Kip1) degradation through direct interaction with JAB1. The pre-S(2) mutant LHBS is suggested to be a potential oncoprotein for HBV-related hepatocellular carcinoma.     

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

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

Low levels of cyclin D and nonfunctional Rb protein affect cdk6 association with cyclin-dependent kinase inhibitor p27(Kip1)

p27(Kip1) associates with cyclin/cdk complexes and inhibiting cdk activity, and overexpression of p27(Kip1) induces G1 arrest. We found that p27(Kip1) overexpression inhibits cdk2 kinase activity, but not cdk6 kinase activity in HeLa cells. The amount of p27(Kip1) associated with cdk2 was significantly higher than that associated with cdk6. cdk6 complexes contained detectable amounts of p27(Kip1) in all human cell lines examined, except in HeLa cells where p27(Kip1) preferentially associated with cdk2. It appears that in HeLa cells overexpressed p27(Kip1) fails to inhibit cdk6 kinase activity because of low binding affinity of cdk6 to p27(Kip1). The low binding affinity is due to a low level of the cdk6/cyclin D complexes. Functional inactivation of pRb has an effect on p27(Kip1) association with cdk6/cyclin D complexes.     

Rat protein tyrosine phosphatase eta suppresses the neoplastic phenotype of retrovirally transformed thyroid cells through the stabilization of p27(Kip1)

The r-PTPeta gene encodes a rat receptor-type protein tyrosine phosphatase whose expression is negatively regulated by neoplastic cell transformation. Here we first demonstrate a dramatic reduction in DEP-1/HPTPeta (the human homolog of r-PTPeta) expression in a panel of human thyroid carcinomas. Subsequently, we show that the reexpression of the r-PTPeta gene in highly malignant rat thyroid cells transformed by retroviruses carrying the v-mos and v-ras-Ki oncogenes suppresses their malignant phenotype. Cell cycle analysis demonstrated that r-PTPeta caused G(1) growth arrest and increased the cyclin-dependent kinase inhibitor p27(Kip1) protein level by reducing the proteasome-dependent degradation rate. We propose that the r-PTPeta tumor suppressor activity is mediated by p27(Kip1) protein stabilization, because suppression of p27(Kip1) protein synthesis using p27-specific antisense oligonucleotides blocked the growth-inhibitory effect induced by r-PTPeta. Furthermore, we provide evidence that in v-mos- or v-ras-Ki-transformed thyroid cells, the p27(Kip1) protein level was regulated by the mitogen-activated protein (MAP) kinase pathway and that r-PTPeta regulated p27(Kip1) stability by preventing v-mos- or v-ras-Ki-induced MAP kinase activation.     

Arginine vasopressin controls p27(Kip1) protein expression by PKC activation and irreversibly inhibits the proliferation of K-Ras-dependent mouse Y1 adrenocortical malignant cells

The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Ras-dependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated β-galactosidase (SA-βGal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27(Kip1) protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27(Kip1) is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27(Kip1), AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP's deleterious effects. These data show that the survival destabilisation of K-Ras-dependent Y1 malignant cells by AVP requires large quantities of the p27(Kip1) protein as well as phosphorylation of the p27(Kip1) protein at both Ser10 and Thr187.     

Cell transformation by the E5/E8 protein of bovine papillomavirus type 4. p27(Kip1), Elevated through increased protein synthesis is sequestered by cyclin D1-CDK4 complexes.

The E5/E8 hydrophobic protein of BPV-4 is, at only 42 residues, the smallest transforming protein identified to date. Transformation of NIH-3T3 cells by E5/E8 correlates with up-regulation of both cyclin A-associated kinase activity and, unusually, p27(Kip1) (p27) but does not rely on changes in cyclin E or cyclin E-CDK2 activity. Here we have examined how p27 is prevented from functioning efficiently as a CDK2 inhibitor, and we investigated the mechanisms used to achieve elevated p27 expression in E5/E8 cells. Our results show that normal subcellular targeting of p27 is not subverted in E5/E8 cells, and p27 retains its ability to inhibit both cyclin E-CDK2 and cyclin A-CDK activities upon release from heat-labile complexes. E5/E8 cells also have elevated levels of cyclins D1 and D3, and high levels of nuclear p27 are tolerated because the inhibitor is sequestered within an elevated pool of cyclin D1-CDK4 complexes, a significant portion of which retain kinase activity. In agreement with this, pRB is constitutively hyperphosphorylated in E5/E8 cells in vivo. The increased steady-state level of p27 is achieved largely through an increased rate of protein synthesis and does not rely on changes in p27 mRNA levels or protein half-life. This is the first report of enhanced p27 synthesis as the main mechanism for increasing protein levels in continuously cycling cells. Our results are consistent with a model in which E5/E8 promotes a coordinated elevation of cyclin D1-CDK4 and p27, as well as cyclin A-associated kinase activity, which act in concert to allow continued proliferation in the absence of mitogens.     

Increased expression of Matrix Metalloproteinase 9 in liver from NZB/W F1 mice received antibody against human parvovirus B19 VP1 unique region protein

Background  

Human parvovirus B19 infection has been postulated to the anti-phospholipid syndrome (APS) in autoimmunity. However, the influence of anti-B19-VP1u antibody in autoimmune diseases is still obscure.     

p27(Kip1) stabilization and G(1) arrest by 1,25-dihydroxyvitamin D(3) in ovarian cancer cells mediated through down-regulation of cyclin E/cyclin-dependent kinase 2 and Skp1-Cullin-F-box protein/Skp2 ubiquitin ligase

p27(Kip1) (p27) is a tumor suppressor whose stability is controlled by proteasome-mediated degradation, a process directed in part by cyclin-dependent kinase 2 (CDK2)-mediated phosphorylation of p27 at Thr(187) and its subsequent interaction with the Skp1-Cullin-F-box protein/Skp2 (Skp2) ubiquitin ligase. The present study shows that 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) arrests ovarian cancer cells in G(1) by stabilizing the p27 protein. 1,25(OH)(2)D(3) initiates a chain of events by decreasing the amounts of cyclin E and cyclin E-associated CDK2 activity. As a result, p27 phosphorylation at Thr(187) and consequently the interaction with Skp2 are decreased. 1,25(OH)(2)D(3) also increases p27 stability by decreasing the abundance of Skp2. It is the combined effect of 1,25(OH)(2)D(3) on both the CDK2-dependent phosphorylation of p27, and thus its affinity for Skp2, and Skp2 expression that dramatically increases the stability of the p27 protein. Similar to its effects in ovarian cancer cells, 1,25(OH)(2)D(3) induces p27 accumulation in wild type mouse embryo fibroblasts and arrests wild type but not p27-null mouse embryo fibroblasts in G(1). Stable expression of Skp2 in OVCAR3 cells diminishes the G(1) arrest and decreases the growth response to 1,25(OH)(2)D(3). Taken together, the results of this study identify p27 as the key mediator of 1,25(OH)(2)D(3)-induced growth suppression in G(1) and show that the hormone achieves this by decreasing the activity of CDK2 and reducing the abundance of Skp2, which act together to degrade p27.     

Helicobacter pylori releases a factor(s) inhibiting cell cycle progression of human gastric cell lines by affecting cyclin E/cdk2 kinase activity and Rb protein phosphorylation through enhanced p27(KIP1) protein expression

Helicobacter pylori, the main cause of chronic gastritis, plays a central role in the etiology of peptic ulcer disease and gastric cancer. In vitro studies have shown that H. pylori increases gastric epithelial cell turnover, thus increasing the risk for the development of neoplastic clones. The mechanisms by which H. pylori promotes perturbation of cell proliferation are not yet elucidated. To investigate whether products released by H. pylori in culture media interfere with cell cycle progression of human gastric epithelial cells, four cell lines (MKN 28, MKN 7, MKN 74, and AGS) were incubated in the presence of H. pylori broth culture filtrate. Cell cycle analysis showed that a H. pylori-released factor(s) significantly inhibited the G1- to S-phase progression of MKN 28 and MKN 7 cell lines, with a reversible, nonlethal mechanism, independent of the expression of VacA, CagA, and/or urease. The cell cycle inhibition occurred concomitantly with an increase in p27(KIP1) protein levels, a reduction in Rb protein phosphorylation on serine residues 807-811, and a significant decrease in cyclin E-associated cdk2 activity. In contrast, the cell cycle progression of MKN 74 and AGS cell lines was not affected by the H. pylori-released factor(s). In normal human fibroblasts, G1-phase cell accumulation was concomitant with the reduction in Rb protein phosphorylation; that, however, appeared to be dependent on p21(WAF1/CIP1) rather than on p27(KIP1) protein. A preliminary characterization showed that the molecular mass of the partially purified cell cycle inhibitory factor(s) was approximately 40 kDa. These results suggest that H. pylori releases a soluble factor(s) that may affect cell cycle progression of gastric epithelial cells through elevated levels of cdk inhibitor p27(KIP1). This factor(s) might act in vivo on noncolonized distant cells, the most proliferating cells of human gastric mucosa.