06-445 Sigma-AldrichAnti-Kip1 (p27) Antibody

p27Kip1 is a cell cycle inhibitor that prevents cyclin dependent kinase (CDK)/cyclin complexes from phosphorylating their targets. p27Kip1 is a known tumor suppressor, as the germline loss of p27Kip1 results in sporadic pituitary formation in aged rodents, and its presence in human cancers is indicative of a poor prognosis. In addition to its role in cancer, loss of p27Kip1 results in regenerative phenotypes in some tissues and maintenance of stem cell pluripotency, suggesting that p27Kip1 inhibitors could be beneficial for tissue regeneration. Because p27Kip1 is an intrinsically disordered protein, identifying direct inhibitors of the p27Kip1 protein is difficult. Therefore, we pursued a high-throughput screening strategy to identify novel p27Kip1 transcriptional inhibitors. We utilized a luciferase reporter plasmid driven by the p27Kip1 promoter to transiently transfect HeLa cells and used cyclohexamide as a positive control for non-specific inhibition. We screened a "bioactive" library consisting of 8,904 (4,359 unique) compounds, of which 830 are Food and Drug Administration (FDA) approved. From this screen, we successfully identified 111 primary hits with inhibitory effect against the promoter of p27Kip1. These hits were further refined using a battery of secondary screens. Here we report four novel p27Kip1 transcriptional inhibitors, and further demonstrate that our most potent hit compound (IC50 = 200 nM) Alsterpaullone 2-cyanoethyl, inhibits p27Kip1 transcription by preventing FoxO3a from binding to the p27Kip1 promoter. This screen represents one of the first attempts to identify inhibitors of p27Kip1 and may prove useful for future tissue regeneration studies.

As erythroid progenitors differentiate into precursors and finally mature red blood cells, lineage-specific genes are induced, and proliferation declines until cell cycle exit. Cul4A encodes a core subunit of a ubiquitin ligase that targets proteins for ubiquitin-mediated degradation, and Cul4A-haploinsufficient mice display hematopoietic dysregulation with fewer multipotential and erythroid-committed progenitors. In this study, stress induced by 5-fluorouracil or phenylhydrazine revealed a delay in the recovery of erythroid progenitors, early precursors, and normal hematocrits in Cul4A(+/-) mice. Conversely, overexpression of Cul4A in a growth factor-dependent, proerythroblast cell line increased proliferation and the proportion of cells in S phase. When these proerythroblasts were induced to terminally differentiate, endogenous Cul4A protein expression declined 3.6-fold. Its enforced expression interfered with erythrocyte maturation and cell cycle exit and, instead, promoted proliferation. Furthermore, p27 normally accumulates during erythroid terminal differentiation, but Cul4A-enforced expression destabilized p27 and attenuated its accumulation. Cul4A and p27 proteins coimmunoprecipitate, indicating that a Cul4A ubiquitin ligase targets p27 for degradation. These findings indicate that a Cul4A ubiquitin ligase positively regulates proliferation by targeting p27 for degradation and that Cul4A down-regulation during terminal erythroid differentiation allows p27 to accumulate and signal cell cycle exit.

Evidence implies that satellite cells could play some limiting role in aged muscle undergoing repair or maintenance of mass, which is of potential clinical concern as this could contribute to sarcopenia. Further, insufficient information is available concerning the cellular mechanisms responsible for the lower rat satellite cell proliferation in old animals. Thus, it was hypothesized that the following proteins would be increased in nuclei of satellite cells from old rat skeletal muscle: the cyclin-dependent kinase (CDK) inhibitors p21(WAF1/CIP1) and p27(Kip1) as well as the transcription factors p53 and Forkhead box, subgroup O1 (FOXO1). In addition, the NAD(+)-dependent histone deacetylase SIRT1, the mammalian ortholog of the yeast SIR2 (silence information regulator 2) and a member of the Sirtuin family, was hypothesized to decrease in satellite cell nuclei of old rats. Old satellite cells (30-months old) exhibited a lesser number of BrdU-positive cells as compared to satellite cells (3-months old) from young growing animals. Western blot analysis demonstrated that nuclei of old satellite cells accumulated the cell cycle inhibitors p21(WAF1/CIP1) and p27(Kip1). In addition, nuclear p53 and FOXO1 proteins were also higher in old satellite cells than in cells from young growing animals. These data indicated both p53/p21(WAF1/CIP1)- and FOXO1/p27(Kip1)-dependent pathways might contribute to the age-associated decrease in satellite cell proliferation. Cytoplasmic manganese superoxide dismutase (MnSOD), a gene driven by FOXO1, was higher in old satellite cells. Unexpectedly, nuclear SIRT1 was also increased in old satellite cells compared with satellite cells from young growing animals. The physiological significance of enhanced nuclear SIRT1 expression in old satellite cells remains elusive at this time. In summary, satellite cells in old rats have nuclear accumulation of proteins inhibiting the cell cycle as compared to young, growing animals.

The insulin-like growth factor I (IGF-I) stimulates muscle satellite cell proliferation. Chakravarthy et al., (2000, J Biol Chem 275:35942-35952.) previously found that IGF-I-stimulated proliferation of primary satellite cells was associated with the activation of phosphatidylinositol 3'-kinase (PI3K)/Akt and the downregulation of a cell-cycle inhibitor p27Kip1. To understand mechanisms by which IGF-I signals the downregulation of p27Kip1 in rat skeletal satellite cells, the role of Forkhead transcription factor FoxO1 in transcriptional activity of p27Kip1 was examined. When primary rat satellite cells were transfected with a p27Kip1 promoter-reporter gene construct, IGF-I (100 ng/ml) inhibited specific p27Kip1 promoter activity. Addition of LY294002, an inhibitor of PI3K, reversed the IGF-I-mediated downregulation of p27Kip1 promoter activity. Co-transfection of wild type (WT) FoxO1 into satellite cells increased p27Kip1 promoter activity in the absence of IGF-I supplementation. Addition of IGF-I reversed the induction of p27Kip1 promoter activity by WT FoxO1. When a mutated FoxO1 (without Thr24, Ser256, and Ser316 Akt phosphorylation sites) was used, IGF-I was no longer able to reverse the FoxO1 induced stimulation of p27Kip1 promoter activity that had been seen when WT FoxO1 was present. When the satellite cells were treated with IGF-I, phosphorylation of Akt-Ser473 and FoxO1-Ser256 was increased. In addition, when the cells were pre-incubated with LY294002 before IGF-I stimulation, the phosphorylation of Akt-Ser473 and FoxO1-Ser256 was inhibited, implying that phosphorylation of Akt and FoxO1 was downstream of IGF-I-induced PI3K signaling. However, IGF-I did not induce phosphorylation of FoxO1 on residues Thr24 and Ser316. These results suggested that IGF-I induced the phosphorylation of Ser256 and inactivated FoxO1 thereby downregulating the activation of the p27Kip1 promoter. Thus, inactivation of FoxO1 by IGF-I plays a critical role in rat skeletal satellite cell proliferation through regulation of p27Kip1 expression.

beta-Catenin is a protein that plays a role in intercellular adhesion as well as in the regulation of gene expression. The latter role of beta-catenin is associated with its oncogenic properties due to the loss of expression or inactivation of the tumor suppressor adenomatous polyposis coli (APC) or mutations in beta-catenin itself. We now demonstrate that another tumor suppressor, PTEN, is also involved in the regulation of nuclear beta-catenin accumulation and T cell factor (TCF) transcriptional activation in an APC-independent manner. We show that nuclear beta-catenin expression is constitutively elevated in PTEN null cells and this elevated expression is reduced upon reexpression of PTEN. TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity. Furthermore, the constitutively elevated expression of cyclin D1, a beta-catenin/TCF-regulated gene, is also suppressed upon reexpression of PTEN. Mechanistically, PTEN increases the phosphorylation of beta-catenin and enhances its rate of degradation. We define a pathway that involves mainly integrin-linked kinase and glycogen synthase kinase 3 in the PTEN-dependent regulation of beta-catenin stability, nuclear beta-catenin expression, and transcriptional activity. Our data indicate that beta-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression.

Interest is growing in methods to extend replicative life span of non-immortalized stem cells. Using the insulin-like growth factor I (IGF-I) transgenic mouse in which the IGF-I transgene is expressed during skeletal muscle development and maturation prior to isolation and during culture of satellite cells (the myogenic stem cells of mature skeletal muscle fibers) as a model system, we elucidated the underlying molecular mechanisms of IGF-I-mediated enhancement of proliferative potential of these cells. Satellite cells from IGF-I transgenic muscles achieved at least five additional population doublings above the maximum that was attained by wild type satellite cells. This IGF-I-induced increase in proliferative potential was mediated via activation of the phosphatidylinositol 3'-kinase/Akt pathway, independent of mitogen-activated protein kinase activity, facilitating G(1)/S cell cycle progression via a down-regulation of p27(Kip1). Adenovirally mediated ectopic overexpression of p27(Kip1) in exponentially growing IGF-I transgenic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin A protein abundance, thereby implicating an important role for p27(Kip1) in promoting satellite cell senescence. These observations provide a more complete dissection of molecular events by which increased local expression of a growth factor in mature skeletal muscle fibers extends replicative life span of primary stem cells than previously known.