Stabilization of p21 (Cip1/WAF1) following Tip60-dependent acetylation is required for p21-mediated DNA damage response

The molecular mechanisms controlling post-translational modifications of p21 have been pursued assiduously in recent years. Here, utilizing mass-spectrometry analysis and site-specific acetyl-p21 antibody, two lysine residues of p21, located at amino-acid sites 161 and 163, were identified as Tip60-mediated acetylation targets for the first time. Detection of adriamycin-induced p21 acetylation, which disappeared after Tip60 depletion with concomitant destabilization of p21 and disruption of G1 arrest, suggested that Tip60-mediated p21 acetylation is necessary for DNA damage-induced cell-cycle regulation. The ability of 2KQ, a mimetic of acetylated p21, to induce cell-cycle arrest and senescence was significantly enhanced in p21 null MEFs compared with those of cells expressing wild-type p21. Together, these observations demonstrate that Tip60-mediated p21 acetylation is a novel and essential regulatory process required for p21-dependent DNA damage-induced cell-cycle arrest.     

Effects of phosphatidylserine on p38 mitogen activated protein kinase,cyclic AMP responding element binding protein and nuclear factor-kappaB activation in resting and activated microglial cells

In the last few years, the interaction between phosphatidylserine (PS), a phospholipid that becomes permanently exposed on the external cell surface in the early phases of apoptosis, and its specific receptor (PtdSerR) has emerged as a crucial event for the engulfing of apoptotic cells and for preventing the acquisition of pro-inflammatory functions by peripheral macrophages. Recently, we demonstrated that PtdSerR is expressed in microglial cultures purified from neonatal rat brain, and that PS-liposomes, used to mimic apoptotic cells, strongly reduce the lipopolysaccharide (LPS)-induced release of inflammatory mediators. Here, we show that in resting microglia, PS-liposomes induce cyclic AMP responding element binding protein (CREB) phosphorylation but do not activate nuclear factor-kappaB (NF-kappaB) and p38 mitogen-activated protein kinase (p38), in line with the non-inflammatory consequences of the recognition and removal of apoptotic cells by macrophages. In LPS-activated microglia, PS-liposomes did not affect NF-kappaB activation but inhibited the phosphorylation of p38 and delayed that of CREB. To our knowledge, this is the first biochemical evidence of the molecular signaling evoked by PS/PtdSerR interaction possibly related to repression of pro-inflammatory activities in microglial cells.     

Sublytic C5b-9 triggers glomerular mesangial cell apoptosis via XAF1 gene activation mediated by p300-dependent IRF-1 acetylation

The apoptosis of glomerular mesangial cells (GMCs) in rat Thy-1 nephritis (Thy-1N), a model of human mesangioproliferative glomerulonephritis (MsPGN), is accompanied by sublytic C5b-9 deposition. However, the mechanism by which sublytic C5b-9 induces GMC apoptosis is unclear. In the present studies, the effect of X-linked inhibitor of apoptosis-associated factor 1 (XAF1) expression on GMC apoptosis and the role of p300 and interferon regulatory factor-1 (IRF-1) in mediating XAF1 gene activation were determined, both in the GMCs induced by sublytic C5b-9 (in vitro) and in the renal tissues of rats with Thy-1N (in vivo). The in vitro studies demonstrated that IRF-1-enhanced XAF1 gene activation and its regulation by p300-mediated IRF-1 acetylation were involved in GMC apoptosis induced by sublytic C5b-9. The element of IRF-1 binding to XAF1 promoter and two acetylated sites of IRF-1 protein were also revealed. In vivo, silence of p300, IRF-1 or XAF1 genes in the renal tissues diminished GMC apoptosis and secondary GMC proliferation as well as urinary protein secretion in Thy-1N rats. Together, these data implicate that sublytic C5b-9 induces the expression of both p300 and IRF-1, as well as p300-dependent IRF-1 acetylation that may contribute to XAF1 gene activation and subsequent GMC apoptosis in Thy-1N rats.     

Peroxisome proliferator-activated receptor gamma suppresses proximal alpha1(I) collagen promoter via inhibition of p300-facilitated NF-I binding to DNA in hepatic stellate cells

Depletion of peroxisome proliferator-activated receptor gamma (PPARgamma) represents one of the key molecular changes that underlie transdifferentiation (activation) of hepatic stellate cells in the genesis of liver fibrosis (Miyahara, T., Schrum, L., Rippe, R., Xiong, S., Yee, H. F., Jr., Motomura, K., Anania, F. A., Willson, T. M., and Tsukamoto, H. (2000) J. Biol. Chem. 275, 35715-35722; Hazra, S., Xiong, S., Wang, J., Rippe, R. A., Krishna, V., Chatterjee, K., and Tsukamoto, H. (2004) J. Biol. Chem. 279, 11392-11401). In support of this notion, ectopic expression of PPARgamma suppresses hepatic stellate cells activation markers, most notably expression of alpha1(I) procollagen. However, the mechanisms underlying this antifibrotic effect are largely unknown. The present study utilized deletion-reporter gene constructs of proximal 2.2-kb alpha1(I) procollagen promoter to demonstrate that a region proximal to -133 bp is where PPARgamma exerts its inhibitory effect. Within this region, two DNase footprints with Sp1 and reverse CCAAT box sites exist. NF-I, but not CCAAT DNA-binding factor/NF-Y, binds to the proximal CCAAT box in hepatic stellate cells. A mutation of this site almost completely abrogates the promoter activity. NF-I mildly but independently stimulates the promoter activity and synergistically promotes Sp1-induced activity. PPARgamma inhibits NF-I binding to the most proximal footprint (-97/-85 bp) and inhibits its transactivity. The former effect is mediated by the ability of PPARgamma to inhibit p300-facilitated NF-I binding to DNA as demonstrated by chromatin immunoprecipitation assay.     

Trace amine-associated receptor 1 (TAAR1) is activated by amiodarone metabolites

Amiodarone (Cordarone, Wyeth-Ayerst Pharmaceuticals) is a clinically available drug used to treat a wide variety of cardiac arrhythmias. We report here the synthesis and characterization of a panel of potential amiodarone metabolites that have significant structural similarity to thyroid hormone and its metabolites the iodothyronamines. Several of these amiodarone derivatives act as specific agonists of the G protein-coupled receptor (GPCR) trace amine-associated receptor 1 (TAAR(1)). This result demonstrates a novel molecular target for amiodarone derivatives with potential clinical significance.     

Exchange protein activated by cyclic AMP (Epac)-mediated induction of suppressor of cytokine signaling 3 (SOCS-3) in vascular endothelial cells

Here, we demonstrate that elevation of intracellular cyclic AMP (cAMP) in vascular endothelial cells (ECs) by either a direct activator of adenylyl cyclase or endogenous cAMP-mobilizing G protein-coupled receptors inhibited the tyrosine phosphorylation of STAT proteins by an interleukin 6 (IL-6) receptor trans-signaling complex (soluble IL-6Ralpha/IL-6). This was associated with the induction of suppressor of cytokine signaling 3 (SOCS-3), a bona fide inhibitor in vivo of gp130, the signal-transducing component of the IL-6 receptor complex. Attenuation of SOCS-3 induction in either ECs or SOCS-3-null murine embryonic fibroblasts abolished the inhibitory effect of cAMP, whereas inhibition of SHP-2, another negative regulator of gp130, was without effect. Interestingly, the inhibition of STAT phosphorylation and SOCS-3 induction did not require cAMP-dependent protein kinase activity but could be recapitulated upon selective activation of the alternative cAMP sensor Epac, a guanine nucleotide exchange factor for Rap1. Consistent with this hypothesis, small interfering RNA-mediated knockdown of Epac1 was sufficient to attenuate both cAMP-mediated SOCS-3 induction and inhibition of STAT phosphorylation, suggesting that Epac activation is both necessary and sufficient to observe these effects. Together, these data argue for the existence of a novel cAMP/Epac/Rap1/SOCS-3 pathway for limiting IL-6 receptor signaling in ECs and illuminate a new mechanism by which cAMP may mediate its potent anti-inflammatory effects.     

Human DNA replication-related element binding factor (hDREF) self-association via hATC domain is necessary for its nuclear accumulation and DNA binding

We previously demonstrated that hDREF, a human homologue of Drosophila DNA replication-related element binding factor (dDREF), is a DNA-binding protein predominantly distributed with granular structures in the nucleus. Here, glutathione S-transferase pulldown and chemical cross-linking assays showed that the carboxyl-terminal hATC domain of hDREF, highly conserved among hAT transposase family members, possesses self-association activity. Immunoprecipitation analyses demonstrated that hDREF self-associates in vivo, dependent on hATC domain. Moreover, analyses using a series of hDREF mutants carrying amino acid substitutions in the hATC domain revealed that conserved hydrophobic amino acids are essential for self-association. Immunofluorescence studies further showed that all hDREF mutants lacking self-association activity failed to accumulate in the nucleus. Self-association-defective hDREF mutants also lost association with endogenous importin beta1. Moreover, electrophoretic gel-mobility shift assays revealed that the mutations completely abolished the DNA binding activity of hDREF. These results suggest that self-association of hDREF via the hATC domain is necessary for its nuclear accumulation and DNA binding. We also found that ZBED4/KIAA0637, another member of the human hAT family, also self-associates, again dependent on the hATC domain, with deletion resulting in loss of efficient nuclear accumulation. Thus, hATC domains of human hAT family members appear to have conserved functions in self-association that are required for nuclear accumulation.