Time- and dose-dependent phosphorylation of Fe65 Ser²⁸⁹ after UV.

<p>(A) GFP-Fe65 S228A was transfected into HEK293 cells 24 hrs prior to treatment with UV (40J/m²). Cells were incubated for 15, 120 and 240 minutes before GFP immunoprecipitation and Western blot analysis with the indicated antibodies. (B) Quantification of p-Fe65x signal in (A) normalised to total GFP-Fe65 in corresponding immunoprecipitate. (C) GFP-Fe65 S228A was transfected into HEK293 cells 24 hrs prior to treatment with 4, 8, 20 or 40 J/m² UV for 2h. After GFP immunoprecipitation, samples were analysed by Western blotting with the indicated antibodies. (D) Data in (C) was quantified as in (B). (E) HEK293 cells were transfected with FLAG-Fe65 S228A for 24h before treatment with DMSO, 5μM AZ20 or 10μM KU55933 for 45min, followed by 40J/m² UV for 2h and GFP immunoprecipitation. Samples were analysed by Western blotting with the indicated antibodies and p-Fe65 quantified relative to FLAG-Fe65 in corresponding immunoprecipitates. Data is the mean and standard deviation from three independent experiments (F) Statistical significance was investigated using a two-tailed paired t-test (* p<0.05). Images are representative of at least three independent experiments.</p

Fe65 Ser²⁸⁹ phosphorylation does not regulate the Fe65-APP interaction.

<p>HEK293 cells were transfected with FLAG-Fe65 or FLAG-Fe65 S289A for 24h before treatment with 40J/m² UV for 2h. Cell extracts were subjected to FLAG immunoprecipitation before western blot analysis with the indicated antibodies. Images are representative of at least three independent experiments.</p

Fe65 Ser²⁸⁹ does not regulate Fe65-APP transcriptional activity.

<p>(A) HEK293 cells were transfected with the indicated plasmids for 24h prior to cell lysis and measurement of both firefly and renilla luciferase activities. To normalise for transfection efficiency the ratio of firefly/renilla luciferase values were calculated (each sample in triplicate). (B) The cellular samples used for the luciferase assay in (A) were analysed by Western blotting to confirm equal expression of both FLAG-Fe65 and FLAG-Fe65 C652F. (C) and (D) HEK293 cells were transfected with the indicated plasmids for 24h prior to luciferase assays and Western blot analysis as in (A) and (B). Data is the mean and standard deviation of triplicate samples and is representative of at least three independent experiments. The significance of the observed changes in relative luciferase activity were investigated using a paired t-test (two-tailed). Levels of significance were defined as follows: p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).</p

Fe65 Is Phosphorylated on Ser²⁸⁹ after UV-Induced DNA Damage

<div><p>Fe65 undergoes a phosphatase-sensitive gel mobility shift after DNA damage, consistent with protein phosphorylation. A recent study identified Ser²²⁸ as a specific site of phosphorylation, targeted by the ATM and ATR protein kinases, with phosphorylation inhibiting the Fe65-dependent transcriptional activity of the amyloid precursor protein (APP). The direct binding of Fe65 to APP not only regulates target gene expression, but also contributes to secretase-mediated processing of APP, producing cytoactive proteolytic fragments including the APP intracellular domain (AICD) and cytotoxic amyloid β (Aβ) peptides. Given that the accumulation of Aβ peptides in neural plaques is a pathological feature of Alzheimer’s disease (AD), it is essential to understand the mechanisms controlling Aβ production. This will aid in the development of potential therapeutic agents that act to limit the deleterious production of Aβ peptides. The Fe65-APP complex has transcriptional activity and the complex is regulated by multiple post-translational modifications and other protein binding partners. In the present study, we have identified Ser²⁸⁹ as a novel site of UV-induced phosphorylation. Interestingly, this phosphorylation was mediated by ATM, rather than ATR, and occurred independently of APP. Neither phosphorylation nor mutation of Ser²⁸⁹ affected the Fe65-APP interaction, though this was markedly decreased after UV treatment, with a concomitant decrease in the protein levels of APP in cells. Using mutagenesis, we demonstrated that Fe65 Ser²⁸⁹ phosphorylation did not affect the transcriptional activity of the Fe65-APP complex, in contrast to the previously described Ser²²⁸ site.</p></div

Fe65 Ser²⁸⁹ phosphorylation occurs independently of the Fe65-APP interaction.

<p>HEK293 cells were transfected with FLAG-Fe65 S228A or FLAG-Fe65 S228A/C652F for 24h before treatment with 40J/m² UV for 2h. Cell extracts were subjected to FLAG immunoprecipitation before Western blot analysis with the indicated antibodies. Images are representative of at least three independent experiments.</p

Sustained Isoprostane E2 Elevation, Inflammation and Fibrosis after Acute Ischaemia-Reperfusion Injury Are Reduced by Pregnane X Receptor Activation

<div><p>Liver grafts donated after cardiac death are increasingly used to expand the donor pool but are prone to ischaemic-type biliary lesions. The anti-inflammatory effects of the activated pregnane X receptor have previously been shown to be beneficial in a number of inflammatory liver conditions. However, its role in reducing peri-portal inflammation and fibrosis following ischaemia-reperfusion injury has not been investigated. Hepatic injury and its response to pregnane X receptor activation was examined after partial hepatic ischaemia-reperfusion injury induced by surgically clamping the left and middle lobar blood vessels in rats. Molecular and pathological changes in the liver were examined over the following 28 days. Ischaemia-reperfusion injury resulted in transient cholestasis associated with microvillar changes in biliary epithelial cell membranes and hepatocellular injury which resolved within days after reperfusion. However, in contrast to chemically-induced acute liver injuries, this was followed by sustained elevation in isoprostane E2, peri-portal inflammation and fibrosis that remained unresolved in the ischaemic reperfused lobe for at least 28 days after clamping. Administration of pregnenolone-16α-carbonitrile—a rodent-specific pregnane X receptor activator—resulted in significant reductions in cholestasis, hepatic injury, ischaemic lobe isoprostane E2 levels, peri-portal inflammation and fibrosis. Hepatic ischaemia-reperfusion injury therefore results in inflammatory and fibrotic changes that persist well beyond the initial ischaemic insult. Drug-mediated activation of the pregnane X receptor reduced these adverse changes in rats, suggesting that the pregnane X receptor is a viable drug target to reduce ischaemic-type biliary lesions in recipients of liver transplants donated after cardiac death.</p></div

IRI results in progressive fibrosis.

<p><b>(A)</b> α-SMA immunohistochemistry—typical views from the indicated treatment group post and time point (upper panels) and quantification of α-SMA immunohistochemistry staining, scale bar represents 100μm. (<b>B)</b> quantification of α-SMA staining. <b>(C)</b> Sirius red staining–typical views from the indicated treatment group post and time point (upper panels) and quantification of Sirius red staining, scale bar represents 100μm. (<b>D</b>) quantification of Sirius red staining. <b>(E-F)</b> qRT-PCR analysis in ischaemic and sham ischaemic lobes. Data are the mean and standard deviation from 3 separate animals at each time point and treatment, *Significantly different compared to sham IRI group, p<0.05.</p

IRI results in persistent inflammatory changes in peri-portal regions and a ductal reaction.

<p><b>(A)</b> H&E stained liver sections of a zone 3 (upper) and zone 1 (lower) region in an IRI lobe (day 1 and day 10 respectively):- BEC, biliary epithelial cell; pv, portal venule; bd, bile ductile; AIC, acute inflammatory cell; H, hepatocyte; F, fibroblast or myofibroblast; hM, haemosiderin-laden macrophage; MG, mononuclear granulocyte. Scale bar represents 100μm. <b>(B-C)</b> Comparison of inflammatory cell counts in peri-portal and centrilobular areas between the IRI and sham IRI groups. (<b>D</b>), cytokeratin 19 (CK-19) staining of liver sections–typical views from the indicated treatment group and time point. Scale bar represents 100μm. E, quantification of CK-19 staining. Data are the mean and standard deviation from 3 separate animals at each time point and treatment, *Significantly different compared to sham IRI group, p<0.05.</p

IRI results in a transient cholestasis and altered biliary physiology.

<p><b>(A)</b> Partial hepatic ischaemia model with bile duct isolation (Left). Schematic representation of groups in both studies (Right). (<b>B)</b> Comparison of bile flow rates (normalised to total body weight). (<b>C)</b> Comparison of serum bile acid concentration between the IRI and sham IRI groups. (<b>D)</b> TEM images comparing BEC microvilli morphology during early (day 1) and late (day 28) reperfusion timepoints. Data are the mean and standard deviation from 3 separate animals at each time point and treatment, *Significantly different compared to sham IRI group, p<0.05.</p

PXR activation reduces IRI-induced ductal reactions and the numbers of fibrogenic cells in the liver.

<p><b>(A, C, E)</b> CK-19, α-SMA and vimentin immunohistochemistry respectively between IRI+PCN and IRI+vehicle groups–typical views from the indicated treatment group at the indicated time point after clamp release (scale bar represents 100μm) with (<b>D, E F)</b> corresponding stain quantification, data are the mean and standard deviation from 5 separate animals at each time point and treatment, *Significantly different compared to IRI + vehicle group, p<0.05.</p