Loss of Prolyl Hydroxylase-1 Protects Against Colitis Through Reduced Epithelial Cell Apoptosis and Increased Barrier Function

Background & Aims Hypoxia inducible factor (HIF) prolyl hydroxylase inhibitors are protective in mouse models of inflammatory bowel disease (IBD). Here, we investigated the therapeutic target(s) and mechanism(s) involved. Methods The effect of genetic deletion of individual HIF-prolyl hydroxylase (PHD) enzymes on the development of dextran sulphate sodium (DSS)induced colitis was examined in mice. Results PHD1-/-, but not PHD2+/- or PHD3-/-, mice were less susceptible to the development of colitis than wild-type controls as determined by weight loss, disease activity, colon histology, neutrophil infiltration, and cytokine expression. Reduced susceptibility of PHD1-/- mice to colitis was associated with increased density of colonic epithelial cells relative to wild-type controls, which was because of decreased levels of apoptosis that resulted in enhanced epithelial barrier function. Furthermore, with the use of cultured epithelial cells it was confirmed that hydroxylase inhibition reversed DSS-induced apoptosis and barrier dysfunction. Finally, PHD1 levels were increased with disease severity in intestinal tissue from patients with IBD and in colonic tissues from DSS-treated mice. Conclusions These results imply a role for PHD1 as a positive regulator of intestinal epithelial cell apoptosis in the inflamed colon. Genetic loss of PHD1 is protective against colitis through decreased epithelial cell apoptosis and consequent enhancement of intestinal epithelial barrier function. Thus, targeted PHD1 inhibition may represent a new therapeutic approach in IBD. © 2010 AGA Institute

Prolyl hydroxylase-1 regulates hepatocyte apoptosis in an NF-kB-dependent manner

Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1(-/-) hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease.status: publishe

A global reference database of crowdsourced cropland data collected using the Geo-Wiki platform

A global reference data set on cropland was collected through a crowdsourcing campaign using the Geo-Wiki crowdsourcing tool. The campaign lasted three weeks, with over 80 participants from around the world reviewing almost 36,000 sample units, focussing on cropland identification. For quality assessment purposes, two additional data sets are provided. The first is a control set of 1,793 sample locations validated by students trained in satellite image interpretation. This data set was used to assess the quality of the crowd as the campaign progressed. The second data set contains 60 expert validations for additional evaluation of the quality of the contributions. All data sets are split into two parts: the first part shows all areas classified as cropland and the second part shows cropland average per location and user. After further processing, the data presented here might be suitable to validate and compare medium and high resolution cropland maps generated using remote sensing. These could also be used to train classification algorithms for developing new maps of land cover and cropland extent

Murine bubblegum orthologue is a microsomal very long-chain acyl-CoA synthetase.

It has been suggested that a gene termed bubblegum (Bgm), encoding an acyl-CoA synthetase, could be involved in the pathogenesis of the inherited neurodegenerative disorder X-ALD (X-linked adrenoleukodystrophy). The precise function of the ALDP (ALD protein) still remains unclear. Aldp deficiency in mammals and Bgm deficiency in Drosophila led to accumulation of VLCFAs (very long-chain fatty acids). As a first step towards studying this interaction in wild-type versus Aldp-deficient mice, we analysed the expression pattern of the murine orthologue of the Bgm gene. In contrast with the ubiquitously expressed Ald gene, Bgm expression is restricted to the tissues that are affected by X-ALD such as brain, testis and adrenals. During mouse brain development, Bgm mRNA was first detected by Northern-blot analysis on embryonic day 18 and increased steadily towards adulthood, whereas the highest level of Ald mRNA was found on embryonic day 12 and decreased gradually during differentiation. Protein fractionation and confocal laser imaging of Bgm-green fluorescent protein fusion proteins revealed a microsomal localization that was different from peroxisomes (where Aldp is detected), endoplasmic reticulum and Golgi. Mouse Bgm showed acyl-CoA synthetase activity towards a VLCFA substrate in addition to LCFAs, and this activity was enriched in the microsomal compartment. Speculating that Bgm expression could be regulated by Ald deficiency, we compared the abundance of Bgm mRNA in wild-type and Ald knockout mice but observed no difference. Although mouse Bgm is capable of activating VLCFA, we conclude that a direct interaction between the mouse Bgm and the Aldp seems unlikely

The oxygen-sensor PHD2 in chondrocytes modifies bone mass by regulating cartilage collagen processing

Laperre K., Fraisl P., Depypere M., Vinckier S., Smets N., Bouillon R., Maes F., Carmeliet P., Carmeliet G., ''The oxygen-sensor PHD2 in chondrocytes modifies bone mass by regulating cartilage collagen processing'', 32nd annual meeting of the American society for bone and mineral research - ASBMR 2010, October 15-19, 2010, Toronto, Ontario, Canada.status: publishe

Very long-chain acyl-CoA synthetase 3: overexpression and growth dependence in lung cancer.

Lung cancer is the leading cause of cancer deaths worldwide. In the United States, only one in six lung cancer patients survives five years after diagnosis. These statistics may improve if new therapeutic targets are identified. We previously reported that an enzyme of fatty acid metabolism, very long-chain acyl-CoA synthetase 3 (ACSVL3), is overexpressed in malignant glioma, and that depleting glioblastoma cells of ACSVL3 diminishes their malignant properties. To determine whether ACSVL3 expression was also increased in lung cancer, we studied tumor histologic sections and lung cancer cell lines. Immunohistochemical analysis of normal human lung showed moderate ACSVL3 expression only in bronchial epithelial cells. In contrast, all of 69 different lung tumors tested, including adeno-, squamous cell, large cell, and small cell carcinomas, had robustly elevated ACSVL3 levels. Western blot analysis of lung cancer cell lines derived from these tumor types also had significantly increased ACSVL3 protein compared to normal bronchial epithelial cells. Decreasing the growth rate of lung cancer cell lines did not change ACSVL3 expression. However, knocking down ACSVL3 expression by RNA interference reduced cell growth rates in culture by 65-76%, and the ability of tumor cells to form colonies in soft agar suspension by 65-80%. We also conducted studies to gain a better understanding of the biochemical properties of human ACSVL3. ACSVL3 mRNA was detected in many human tissues, but the expression pattern differed somewhat from that of the mouse. The enzyme activated long- and very long-chain saturated fatty acid substrates, as well as long-chain mono- and polyunsaturated fatty acids to their respective coenzyme A derivatives. Endogenous human ACSVL3 protein was found in a punctate subcellular compartment that partially colocalized with mitochondria as determined by immunofluorescence microscopy and subcellular fractionation. From these studies, we conclude that ACSVL3 is a promising new therapeutic target in lung cancer

Fatty acid composition of control and ACSVL3-depleted lung cancer cell lines.

<p>Cells were grown to near confluence prior to harvest. Lipids were extracted, hydrolyzed, and FA converted to their pentafluoobenzyl bromide derivatives prior to quantitation by GC-MS as described in Methods. For H460, three independent knockdown clones were averaged; for EKVX, two independent knockdown clones were averaged. Results are presented as percent of total FA (± SEM for H460 knockdown).</p

ACSVL3 knockdown in lung cancer cell lines.

<p>The short hairpin-producing vector pSilencer (Ambion) was used to generate several lung cancer cell lines with decreased ACSVL3 expression as described in methods. Clonal lines of H460, H82, A549, and EKVX lung cancer cells with stable knockdown (KD) of ACSVL3 were selected. Control lines stably harbor a plasmid containing a scrambled sequence that does not target any human or rodent mRNA. (<i>A</i>) ACSVL3 expression in control and KD cells was assessed by Western blotting. (<i>B</i>) Gel-Pro Analyzer 4.0 software was used to quantitate the Western blot signals in (A). Expression of ACSVL3 relative to β-actin was calculated for each pair of control and KD cell lines. Control cell line ratios were arbitrarily set to 100 and the relative, normalized expression of ACSVL3 in KD cells was calculated. White bars, control cells; grey bars, KD cells.</p

ACSVL3 localization in HepG2 cell subcellular fractions.

<p>HepG2 cells were fractionated into nuclear (N), mitochondrial (M), light mitochondrial (L), microsomal (P), and cytosolic (S) fractions by differential centrifugation, and a total mitochondrial (ML) fraction was further separated into purified mitochondrial (Mito) and mitochondria-associated membrane (MAM) fractions, as described in Methods. Each lane was loaded with ∼30 µg protein. Western blot analyses of the same membrane were performed using antibodies against ACSVL3, the mitochondrial marker Mn-superoxide dismutase (MnSOD), and the MAM marker phosphatidylethanolamine N-methyltransferase (PEMT).</p