Identification of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) as a novel downstream target of phosphatidylinositol 3-kinase/AKT/GSK-3beta pathway

The signaling pathway of phosphatidylinositol 3-kinase (PI3K)/AKT, which is involved in cell survival, proliferation, and growth, has become a major focus in targeting cancer therapeutics. Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) was previously identified as a gene induced by several anti-tumorigenic compounds including nonsteroidal anti-inflammatory drugs, peroxisome proliferator-activated receptor gamma ligands, and dietary compounds. NAG-1 has been shown to exhibit anti-tumorigenic and/or pro-apoptotic activities in vivo and in vitro. In this report, we showed a PI3K/AKT/glycogen synthase kinase-3beta (GSK-3beta) pathway regulates NAG-1 expression in human colorectal cancer cells as assessed by the inhibition of PI3K, AKT, and GSK-3beta. PI3K inhibition by LY294002 showed an increase in NAG-1 protein and mRNA expression, and 1l-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (AKT inhibitor) also induced NAG-1 expression. LY294002 caused increased apoptosis, cell cycle, and cell growth arrest in HCT-116 cells. Inhibition of GSK-3beta, which is negatively regulated by AKT, using AR-A014418 and lithium chloride completely abolished LY294002-induced NAG-1 expression as well as the NAG-1 promoter activity. Furthermore, the down-regulation of GSK-3 gene using small interference RNA resulted in a decline of the NAG-1 expression in the presence of LY294002. These data suggest that expression of NAG-1 is regulated by PI3K/AKT/GSK-3beta pathway in HCT-116 cells and may provide a further understanding of the important role of PI3K/AKT/GSK-3beta pathway in tumorigenesis.     

Stimulation of ultraviolet-induced apoptosis of human fibroblast UVr-1 cells by tyrosine kinase inhibitors

Damnacanthal is an anthraquinone compound isolated from the root of Morinda citrifolia and was reported to have a potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptor. In the present study, we have examined the effects of damnacanthal on ultraviolet ray-induced apoptosis in ultraviolet-resistant human UVr-1 cells. When the cells were treated with damnacanthal prior to ultraviolet irradiation, DNA fragmentation was more pronounced as compared to the case of ultraviolet irradiation alone. The other tyrosine kinase inhibitors, herbimycin A and genistein, also caused similar effects on ultraviolet-induced apoptosis but to a lesser extent. Serine/threonine kinase inhibitors, K252a, staurosporine and GF109203X, rather suppressed the ultraviolet-induced DNA cleavage. Immunoblot analysis showed that pretreatment with damnacanthal followed by ultraviolet irradiation increased the levels of phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases. However, the other tyrosine kinase inhibitors did not increase the phosphorylation of extracellular signal-regulated kinases but stimulated phosphorylation of stress-activated protein kinases. Consequently, the ultraviolet-induced concurrent increase in both phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases after pretreatment with damnacanthal might be characteristically related to the stimulatory effect of damnacanthal on ultraviolet-induced apoptosis.     

3'UTR elements inhibit Ras-induced C/EBPβ post-translational activation and senescence in tumour cells

C/EBPβ is an auto-repressed protein that becomes post-translationally activated by Ras-MEK-ERK signalling. C/EBPβ is required for oncogene-induced senescence (OIS) of primary fibroblasts, but also displays pro-oncogenic functions in many tumour cells. Here, we show that C/EBPβ activation by H-Ras(V12) is suppressed in immortalized/transformed cells, but not in primary cells, by its 3' untranslated region (3'UTR). 3'UTR sequences inhibited Ras-induced cytostatic activity of C/EBPβ, DNA binding, transactivation, phosphorylation, and homodimerization, without significantly affecting protein expression. The 3'UTR suppressed induction of senescence-associated C/EBPβ target genes, while promoting expression of genes linked to cancers and TGFβ signalling. An AU-rich element (ARE) and its cognate RNA-binding protein, HuR, were required for 3'UTR inhibition. These components also excluded the Cebpb mRNA from a perinuclear cytoplasmic region that contains activated ERK1/2, indicating that the site of C/EBPβ translation controls de-repression by Ras signalling. Notably, 3'UTR inhibition and Cebpb mRNA compartmentalization were absent in primary fibroblasts, allowing Ras-induced C/EBPβ activation and OIS to proceed. Our findings reveal a novel mechanism whereby non-coding mRNA sequences selectively regulate C/EBPβ activity and suppress its anti-oncogenic functions.     

Neudesin, an extracellular heme-binding protein, suppresses adipogenesis in 3T3-L1 cells via the MAPK cascade

Adult mice abundantly express neudesin, an extracellular heme-binding protein with neurotrophic activity, in white adipose tissues. At the early stage of adipocyte differentiation during adipogenesis, however, the expression of neudesin decreased transiently. Neudesin-hemin significantly suppressed adipogenesis in 3T3-L1 cells. The knockdown of neudesin by RNA interference markedly promoted adipogenesis in 3T3-L1 cells and decreased MAPK activation during adipocyte differentiation. The addition or knockdown of neudesin affected the expression of C/EBPα and PPARγ but not of C/EBPβ. These findings suggest that neudesin plays a critical role in the early stage of adipocyte differentiation in which C/EBPβ induces PPARγ and C/EBPα expressions, by controlling the MAPK pathway.     

CCAAT/enhancer-binding protein-β participates in oxidized LDL-enhanced proliferation in 3T3-L1 cells

Increased circulating oxidized LDL (oxLDL) have been found in obese subjects. Obesity is characterized by an excess of fat mass resulting from an increase in adipocyte number and size. The generation of new adipocytes is a tightly controlled process where multiple factors acting in a signaling cascade follow a precise temporal expression pattern; oxLDL appear to have a role in the impairment of this process. The purpose of this study was to examine the effects of oxLDL on the mechanisms involved in the proliferative stage of the differentiation process in 3T3-L1 cells. After hormonal induction, 3T3-L1 cells undergo approximately two rounds of mitotic clonal expansion (MCE), a process required for adipogenesis. CCAAT/enhancer-binding protein β (C/EBPβ) is immediately expressed after induction, and plays a crucial role in MCE, but its expression must decrease to allow preadipocytes to mature into adipocytes. We found that, in the presence of stimuli to differentiate, oxLDL induced a higher proliferation rate in this cell line, associated with a sustained up-regulation of C/EBPβ, which remained activated inside the nucleus for several days. RNAi-mediated knockdown of C/EBPβ 24 h after oxLDL treatment counteracted the increase in proliferation rate. Both C/EBPβ expression and proliferation processes appear to be influenced by cAMP/protein kinase A (PKA) and extracellular signal-regulated kinases1/2 (ERK1/2) pathways. OxLDL treatment led to increased levels of cAMP, and to a strong, prolonged phosphorylation of ERK1/2 and C/EBPβ. The addition of cAMP and PKA inhibitors, SQ22536 and H-89, respectively, reduced proliferation only in oxLDL-treated cells, whereas the addition of ERK1/2 inhibitor U0126 blocked proliferation in both control and oxLDL-treated cells. C/EBPβ nuclear expression and DNA-binding activity were reduced by U0126, under all tested conditions. These findings show that the altered expression pattern of C/EBPβ is involved in the increase in the number of proliferating cells induced by oxLDL, in hormone-stimulated 3T3-L1 cells.