Koozi Matuoka

Transcriptional regulation of cellular ageing by the CCAAT box-binding factor CBF/NF-Y

Cellular ageing is a systematic process affecting the entirety of cell structure and function. Since changes in gene expression are extensive and global during ageing, involvement of general transcription regulators in the phenomenon is likely. Here, we focus on NF-Y, the major CCAAT box-binding factor, which exerts differential regulation on a wide variety of genes through its interaction with the CCAAT box present in as many as 25% of the eukaryotic genes. When a cell ages, senescing signals arise, typically through DNA damage due to oxidative stress or telomere shortening, and are transduced to proteins such as p53, retinoblastoma protein, and phosphatidylinositol 3-kinase. Among them, activated p53 family proteins suppress the function of NF-Y and thereby downregulate a set of cell cycle-related genes, including E2F1, which further leads to downregulation of E2F-regulated genes and cell cycle arrest. The p53 family also induces other ageing phenotypes such as morphological alterations and senescence-associated beta-galactosidase (SA-gal) presumably by upregulation of some genes through NF-Y suppression. In fact, the activities of NF-Y and E2F decrease during ageing and a dominant negative NF-YA induces SA-gal. Based on these observations, NF-Y appears to play an important role in the process of cellular ageing.

A positive role of phosphatidylinositol 3-kinase in aging phenotype expression in cultured human diploid fibroblasts

In order to detect the role that phosphatidylinositol 3-kinase (PI3K) plays in the aging of human diploid fibroblasts, we analyzed cellular inositol phospholipids and expression of PI3Ks. In aged cells a decrease in phosphatidylinositol 3,4-bisphosphate (PI3,4P(2)) was notable, while phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) decreased slightly. On the other hand, the messages of PI3K IIalpha, Vps34, and p110delta decreased and that of PI3K IIbeta increased during aging. These changes might relate to the aging phenomena, with the PI3K subspecies functioning differentially. Consistently, a PI3K inhibitor LY294002 greatly suppressed enlargement and flattening of cell body and nucleus as well as cell proliferation, both phenotypes being typical of aged cells. An oxidative stress, pulse exposure to hydrogen peroxide (H(2)O(2)), induced these senescent cell-like phenotypes, which LY294002 was also able to abolish. Upon examining three different cell systems (HL-60, N1E-115, and PC-12 cells) we found clear parallelism in a cellular event between the dependence on a PI3K activity and the sensitivity to H(2)O(2). On the analogy of these relationships, we could hypothesize that expression of an aging phenotype such as the morphogenesis is positively promoted by some PI3K subspecies, if such a phenotype as cell cycling is negatively affected by attenuation of another PI3K function in the course of cellular aging.

Possible role of subunit A of nuclear factor Y (NF-YA) in normal human diploid fibroblasts during senescence.

NF-Y, a heterotrimeric CCAAT binding protein, may have a role in regulating some G1/S genes whose expressions are attenuated during replicative senescence [Matuoka and Chen (1999) Exp Cell Res 253: 365–371]. The hallmark of replicative senescence is the loss of dividing potential. Hence, attenuation of G1/S gene expressions may be causally related to aging. To understand how NF-Y is involved in regulating G1/S genes during replicative senescence, we have examined the expressions of three NF-Y subunit genes in human IMR-90 cells over the entire course of their life-span. The mRNA levels of NF-YA, B, and C did not show any age-dependent change. In contrast, the protein level of NF-YA exhibited a significant and progressive decrease during cell senescence. Cross-linking experiments indicated that NF-Y may interact with proteins such as GCN5 and P/CAF. Co-transfection of cells with plasmid encoding NF-YA protein enhanced the expression of reporter gene fused with G1/S gene promoter that contains NF-Y sites. In contrast, co-transfection with plasmid encoding the dominant negative NF-YA mutant suppressed the expression of the reporter genes. Transient transfection of human cells with dominant negative NF-YA mutant could lead to an increase in neutral β-galactosidase activity, a marker of cell senescence. These results support the view that NF-Y may have a role in cell senescence.

Cytoskeletal reorganization induced by insulin: involvement of Grb2/Ash, Ras and phosphatidylinositol 3-kinase signalling

Background:  Cytoskeletal reorganization is important for a wide variety of insulin‐mediated biological actions, including cell growth, migration and metabolism, but the intracellular signalling pathways leading to insulin‐induced cytoskeletal reorganization have largely been unknown. We therefore investigated the involvement of Grb2/Ash‐Ras and phosphatidylinositol (PI) 3‐kinase in the insulin‐induced morphological changes in fibroblasts over‐expressing human insulin receptors (HIRcB cells).

Telomerase positive human diploid fibroblasts are resistant to replicative senescence but not premature senescence induced by chemical reagents

Human diploid fibroblasts in tissue culture undergo replicative senescence after a finite number of divisions that is characterized by a permanent loss of their dividing potential. However, senescence-like phenotypes, including growth cessation, morphological changes, and appearance of senescence-associated β-galactosidae (SA-gal) activity, can be induced by treating early passage cells with C6-ceramide, H2O2, LY294002, or trichostatin A. While there is convincing evidence that telomere shortening is causally related to replicative senescence, the role of telomere shortening in the chemical-induced premature senescence is unclear. Here we employed a normal human BJ cell strain and its telomerase-transfected counterpart, termed BJ-T cells, to examine whether active telomerase in BJ-T can block or delay the premature senescence induced by various chemicals and, if not, whether telomere shortening still occurs. We found that, although all four chemicals tested could induce growth arrest, and in some cases SA-gal activity, in both BJ and BJ-T cells, only H2O2 clearly caused an irreversible loss of dividing potential. H2O2 treatment did not inhibit the cellular telomerase activity, nor did it cause any appreciable telomere shortening in BJ-T cells. These results suggest that oxidative stress and other chemical reagents can target at sites unrelated to the telomere-associated clocking mechanism. Alternatively these chemicals may bypass the telomere length maintenance machinery and target at its downstream sites.

Downregulated expression of the signaling molecules Nck, c-Crk, Grb2/Ash, PI 3-kinase p110α and WRN during fibroblast aging in vitro

An RT-PCR analysis was performed to examine changes in intracellular signal transducing molecules during in-vitro aging of human fibroblasts. Expression of Nck, c-Crk, Grb2/Ash, phosphoinositide (PI) 3-kinase p110 alpha and Werners syndrome gene product WRN was noticeably reduced in late passage cells, showing a concurrent downregulation of a set of signaling molecules accompanying aging.