The capacity of DNA damaging agents to induce apoptosis is regulated by target gene induction by p53. We found that p53 targeted MDM2 in cells in which DNA repair was occurring, but persistent DNA damage induced by chemotherapy led p53 to selectively target PTEN. High dose chemotherapy induced the phosphorylation of p53 on serine 46, whereas low dose chemotherapy did not. A nonphosphorylatable serine 46 to alanine p53 mutant (S46A) targeted the MDM2 promoter in preference to that for PTEN. A serine 46 to aspartate mutant (S46D, a phosphorylation mimic) targeted PTEN in preference to MDM2. These observations show that phosphorylation of serine 46 in p53 is sufficient for it to induce the PTEN (phosphatase and tensin homolog deleted on chromosome ten) tumor suppressor protein in preference to MDM2. S46A induced significantly less cell death than the S46D in cells. The phosphorylation-induced change of p53 promoter targeting suppresses the induction of MDM2 and the formation of the autoregulatory feedback loop. Induction of PTEN by p53 followed by expression of PTEN inhibits AKT-induced translocation of MDM2 into the nucleus and sustains p53 function. The protection of p53 from MDM2 by PTEN and the damage-induced activation of PTEN by phosphorylated p53 leads to the formation of an apoptotic amplification cycle in which p53 and PTEN coordinately increase cellular apoptosis.

The NF-B family of transcription factors plays a fundamental role in development, maintenance of the immune system, and cell viability (1-3). NF-B is composed of heterodimers of DNA-binding subunits (p50 and p52) and subunits with transcriptional activity (p65 (RelA), RelB, or c-Rel). In unstimulated cells, binary complexes of these subunits are restricted to the cytoplasm by interaction with members of a family of inhibitory proteins, inhibitors of B (IBs) 1 (4, 5). In response to extracellular stimuli such as cytokines or UV radiation, IB proteins are phosphorylated, polyubiquitinated, and then degraded by the 26 S proteasome (6 -13). Dissociation from IBs unmasks the nuclear localization sequence of NF-B, permitting it to move into the nucleus, bind the promoters of target genes, and alter gene expression and cell function (10,13,14). The demonstration that phosphorylation of IB proteins initiates events necessary for activation of NF-B led to the discovery of IB kinase (IKK) complexes composed of IKK␣, IKK␤, and IKK␥ (NEMO) (15)(16)(17)(18)(19). IKK␣ and IKK␤ are serine-threonine kinases, and IKK␥ is a scaffolding protein essential for the function of IKK␣ and IKK␤. IKK␣ and IKK␤ share a high degree of amino acid homology and domain organization. The kinases are composed of an N-terminal kinase domain, a leucine zipper that facilitates homo-and heterodimerization, and a helix-loop-helix domain (20). IKK␣ and IKK␤ can be activated by diverse kinases, among which are NF-B-inducing kinase (NIK), MEKK1, Cot, NF-Bactivating kinase (NAK/TBK), protein kinases C␤ and C␦, and MEKK3 (21-28). However, interest remains sustained in identifying other kinases that affect IKK complexes and NF-B activity. One of these is the Akt serine-threonine kinase, a downstream target for activated phosphatidylinositol 3-kinase (PI 3-kinase) (29 -32). Akt is activated by mitogens and cytokines that function as survival factors. Akt mediates its functions by phosphorylating substrates that decrease the activity of pro-apoptotic proteins or increase the activity of anti-apoptotic proteins (32)(33)(34)(35)(36)(37)(38)(39)(40)(41).Akt may affect NF-B through multiple mechanisms. We demonstrated previously that TNF activates Akt, which phosphorylates and activates IKK␣, thus promoting NF-B function (42). TNF and interleukin-1 can also increase the transactivation potential of the RelA/p65 subunit of NF-B through a mechanism in which Akt has been implicated (43-45). PI 3-kinase activated by phorbol esters or lipopolysaccharide and PI 3-kinase/Akt signaling induced by signaling through CD40, interleukin-1, or G protein-coupled receptors activates [46][47][48]. However, PI 3-kinase/Akt signaling induced by TNF in human umbilical vein endothelial cells inhibits apoptosis without playing a significant role in activation of NF-B (49). Furthermore, Akt can activate a member of the mitogenactivated protein kinase kinase kinase (MAP3K) family, Cot, and indirectly affect IKK activity and NF-B (50). Thus, PI 3-kinase/Akt signaling is upstream of diver...

Oxymatrine is a traditional Chinese herbal product that exhibits anti-inflammatory effects in models of heart, brain and liver injury. We investigated the impact of oxymatrine in an acute model of intestinal injury and inflammation. Oxymatrine significantly decreased LPS-induced NF-κB-driven luciferase activity, correlating with diminished induction of Cxcl2, Tnfα and Il6 mRNA expression in rat IEC-6 and murine BMDC. Although oxymatrine decreased LPS-induced p65 nuclear translocation and binding to the Cxcl2 gene promoter, this effect was independent of IκBα degradation/phosphorylation. DSS-induced weight loss and histological damage were ameliorated in oxymatrine-treated C57BL/6-WT-mice. While this effect correlated with reduced colonic Il6 and Il1β mRNA accumulation, global NF-κB activity as measured in NF-κBEGFP mice was unaffected. Our data demonstrate that oxymatrine reduces LPS-induced NF-κB nuclear translocation and activity independently of IκBα status, prevents intestinal inflammation through blockade of inflammatory signaling and ameliorates overall intestinal inflammation in vivo.

BackgroundTheaflavins including theaflavin (TF), theaflavin-3-gallate (TF3G), theaflavin-3′-gallate (TF3′G), and theaflavin-3,3′-digallate (TFDG), are the most important bioactive polyphenols in black tea. Because of their poor systemic bioavailability, it is still unclear how these compounds can exert their biological functions. The objective of this study is to identify the microbial metabolites of theaflavins in mice and in humans.Methods and FindingsIn the present study, we gavaged specific pathogen free (SPF) mice and germ free (GF) mice with 200 mg/kg TFDG and identified TF, TF3G, TF3′G, and gallic acid as the major fecal metabolites of TFDG in SPF mice. These metabolites were absent in TFDG- gavaged GF mice. The microbial bioconversion of TFDG, TF3G, and TF3′G was also investigated in vitro using fecal slurries collected from three healthy human subjects. Our results indicate that TFDG is metabolized to TF, TF3G, TF3′G, gallic acid, and pyrogallol by human microbiota. Moreover, both TF3G and TF3′G are metabolized to TF, gallic acid, and pyrogallol by human microbiota. Importantly, we observed interindividual differences on the metabolism rate of gallic acid to pyrogallol among the three human subjects. In addition, we demonstrated that Lactobacillus plantarum 299v and Bacillus subtilis have the capacity to metabolize TFDG.ConclusionsThe microbiota is important for the metabolism of theaflavins in both mice and humans. The in vivo functional impact of microbiota-generated theaflavins-derived metabolites is worthwhile of further study.