SUMMARY Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. COPD is caused by chronic exposure to cigarette smoke and/or other environmental pollutants that are believed to induce reactive oxygen species (ROS) that gradually disrupt signalling pathways responsible for maintaining lung integrity. Here we identify the antioxidant protein sestrin-2 (SESN2) as a repressor of PDGFRβ signalling, and PDGFRβ signalling as an upstream regulator of alveolar maintenance programmes. In mice, the mutational inactivation of Sesn2 prevents the development of cigarette-smoke-induced pulmonary emphysema by upregulating PDGFRβ expression via a selective accumulation of intracellular superoxide anions (O2−). We also show that SESN2 is overexpressed and PDGFRβ downregulated in the emphysematous lungs of individuals with COPD and to a lesser extent in human lungs of habitual smokers without COPD, implicating a negative SESN2-PDGFRβ interrelationship in the pathogenesis of COPD. Taken together, our results imply that SESN2 could serve as both a biomarker and as a drug target in the clinical management of COPD.

AIMS:Neuropathic pain is a chronic debilitating disease that is often unresponsive to currently available treatments. Emerging lines of evidence indicate that reactive oxygen species (ROS) are required for the development and maintenance of neuropathic pain. However, little is known about endogenous mechanisms that neutralize the pain-relevant effects of ROS. In the present study, we tested whether the stress-responsive antioxidant protein Sestrin 2 (Sesn2) blocks the ROS-induced neuropathic pain processing in vivo.RESULTS:We observed that Sesn2 mRNA and protein expression was up-regulated in peripheral nerves after spared nerve injury, a well-characterized model of neuropathic pain. Sesn2 knockout (Sesn2(-/-)) mice exhibited considerably increased late-phase neuropathic pain behavior, while their behavior in acute nociceptive and in inflammatory pain models remained unaffected. The exacerbated neuropathic pain behavior of Sesn2(-/-) mice was associated with elevated ROS levels and an enhanced activating transcription factor 3 up-regulation in sensory neurons, and it was reversed by the ROS scavenger N-tert-Butyl-α-phenylnitrone. In contrast, administration of the ROS donor tert-butyl hydroperoxide induced a prolonged pain behavior in naive Sesn2(-/-) mice.INNOVATION:We show that the antioxidant function of Sesn2 limits neuropathic pain processing in vivo.CONCLUSION:Sesn2 controls ROS-dependent neuropathic pain signaling after peripheral nerve injury and may, thus, provide a potential new target for the clinical management of chronic neuropathic pain conditions.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Cigarette smoking has been identified as one of the major risk factors and several predisposing genetic factors have been implicated in the pathogenesis of COPD, including a single nucleotide polymorphism (SNP) in the latent transforming growth factor (TGF)-beta binding protein 4 (Ltbp4)-encoding gene. Consistent with this finding, mice with a null mutation of the short splice variant of Ltbp4 (Ltbp4S) develop pulmonary emphysema that is reminiscent of COPD. Here, we report that the mutational inactivation of the antioxidant protein sestrin 2 (sesn2) partially rescues the emphysema phenotype of Ltbp4S mice and is associated with activation of the TGF-beta and mammalian target of rapamycin (mTOR) signal transduction pathways. The results suggest that sesn2 could be clinically relevant to patients with COPD who might benefit from antagonists of sestrin function.

The blood-brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/beta-catenin (beta-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of beta-cat in vivo enhances barrier maturation, whereas inactivation of beta-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of beta-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of beta-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of beta-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown.

In the central nervous system, a constant microenvironment required for neuronal cell activity is maintained by the blood-brain barrier (BBB). The BBB is formed by the brain microvascular endothelial cells (BMEC), which are sealed by tight junctions (TJ). To identify genes that are differentially expressed in BMEC compared with peripheral endothelial cells, we constructed a subtractive cDNA library from porcine BMEC (pBMEC) and aortic endothelial cells (AOEC). Screening the library for differentially expressed genes yielded 26 BMEC-specific transcripts, such as solute carrier family 35 member F2 (SLC35F2), ADP-ribosylation factor-like 5B (ARL5B), TSC22 domain family member 1 (TSC22D1), integral membrane protein 2A (ITM2A), and epithelial membrane protein 1 (EMP1). In this study, we show that EMP1 transcript is enriched in pBMEC compared with brain tissue and that EMP1 protein colocalizes with the TJ protein occludin in mouse BMEC by coimmunoprecipitation and in rat brain vessels by immunohistochemistry. Epithelial membrane protein 1 expression was transiently induced in laser-capture microdissected rat brain vessels after a 20-min global cerebral ischemia, in parallel with the loss of occludin immunoreactivity. The study identifies EMP1 as a novel TJ-associated protein of the BBB and suggests its potential role in the regulation of the BBB function in cerebral ischemia.

Transforming growth factor-betas (TGF-betas) are multifunctional growth factors that are secreted as inactive (latent) precursors in large protein complexes. These complexes include the latency-associated propeptide (LAP) and a latent transforming growth factor-beta binding protein (LTBP). Four isoforms of LTBPs (LTBP-1-LTBP-4) have been cloned and are believed to be structural components of connective tissue microfibrils and local regulators of TGF-beta tissue deposition and signaling. By using a gene trap strategy that selects for integrations into genes induced transiently during early mouse development, we have disrupted the mouse homolog of the human LTBP-4 gene. Mice homozygous for the disrupted allele develop severe pulmonary emphysema, cardiomyopathy, and colorectal cancer. These highly tissue-specific abnormalities are associated with profound defects in the elastic fiber structure and with a reduced deposition of TGF-beta in the extracellular space. As a consequence, epithelial cells have reduced levels of phosphorylated Smad2 proteins, overexpress c-myc, and undergo uncontrolled proliferation. This phenotype supports the predicted dual role of LTBP-4 as a structural component of the extracellular matrix and as a local regulator of TGF-beta tissue deposition and signaling.

The RUNX1 gene on human chromosome 21q22.12 belongs to the ‘runt domain’ gene family of transcription factors (also known as AML/CBFA/PEBP2α). RUNX1 is a key regulator of hematopoiesis and a frequent target of leukemia associated chromosomal translocations. Here we present a detailed analysis of the RUNX1 locus based on its complete genomic sequence. RUNX1 spans 260 kb and its expression is regulated through two distinct promoter regions, that are 160 kb apart. A very large CpG island complex marks the proximal promoter (promoter-2), and an additional CpG island is located at the 3′ end of the gene. Hitherto, 12 different alternatively spliced RUNX1 cDNAs have been identified. Genomic sequence analysis of intron/exon boundaries of these cDNAs has shown that all consist of properly spliced authentic coding regions. This indicates that the large repertoire of RUNX1 proteins, ranging in size between 20–52 kDa, are generated through usage of alternatively spliced exons some of which contain in frame stop codons. The gene’s introns are largely depleted of repetitive sequences, especially of the LINE1 family. The RUNX1 locus marks the transition from a ~1 Mb of gene-poor region containing only pseudogenes, to a gene-rich region containing several functional genes. A search for RUNX1 sequences that may be involved in the high frequency of chromosomal translocations revealed that a 555 bp long segment originating in chromosome 11 FLI1 gene was transposed into RUNX1 intron 4.1. This intron harbors the t(8;21) and t(3;21) chromosomal breakpoints involved in acute myeloid leukemia. Interestingly, the FLI1 homologous sequence contains a breakpoint of the t(11;22) translocation associated with Ewing's tumors, and may have a similar function in RUNX1.

5-Oxo-L-prolinase (5-OPase) catalyses the hydrolysis of 5-oxo-L-proline to glutamate with concomitant stoichiometric cleavage of ATP to ADP, a reaction which is known to be part of the gamma-glutamyl cycle-an interrelated series of reactions involved in the synthesis and metabolism of glutathione. As recent studies indicate, this cyclic pathway plays a crucial role in the regulation of amino acid transport. Apparently, the intermediate product 5-oxo-L-proline functions as a second messenger molecule that upregulates the activity of certain amino acid transport systems. Thus, the degradation of 5-oxo-L-proline by 5-OPase leads to the downregulation of this stimulus. In this study, a new sensitive fluorimetric assay for 5-OPase activity was established which is based on the derivatization of glutamate with o-phthaldialdehyde in the presence of thiols and subsequent separation of the products by HPLC. The method is suitable for the screening of chromatography fractions as well as for the determination of the kinetic parameters Km and Vmax of purified 5-OPase. Additionally, it can be used for the measurement of enzyme activity in crude cell extracts and evaluation of tissue distribution.

5-0xo-L-prolinase (E.C. 3.5.2.9) was purified to homogeneity from porcine kidney. The molecular weight of one subunit was estimated to 135 kDa by SDS-PAGE. N-terrninal sequencing was not successful, leading to the conclusion that the enzyme is N-terminal blocked. Partial sequence data from a chymotryptic peptide showed strong similarities to the rat enzyme.

Subtractive cloning procedures led to the identification of a variety of transcripts expressed in mammalian brain. However, little is known about the encoded proteins and the regulation of gene expression. Here, we describe the isolation and characterisation of a single-copy gene (83.5) of 21.7 kb which is specifically expressed in porcine brain. In situ hybridisation and immunohistochemistry experiments showed a distinct pattern of gene expression in neuronal cell types in different parts of the brain. The gene contains two mini exons, confirming neural-specific expression. cDNA cloning experiments revealed two species of mRNA differing in their 5'-regions. These transcripts are generated by two distinct transcription start sites that are under the control of different potential promoter regions as shown by primer-extension experiments. The amino acid sequences of the deduced proteins predict that one mRNA species encodes a novel type-I transmembrane protein, whereas the other transcript encodes only a part of its cytoplasmic domain. In Western-blot experiments, we detected two proteins of the predicted size and cellular localisation in porcine brain. The precise function of these proteins remains to be determined. However, our findings suggest that they may be generated by alternative promoter usage, leading to the expression of a membrane protein and its truncated cytoplasmic isoform.

We have determined the genomic sequence of a porcine protein kinase (PPK) gene, including 1,844 bp upstream of the transcription initiation site. The gene spans over 19 kb and consists of 18 exons and 17 introns. The 5' regulatory region contains a characteristic heat shock element in the first intron, a weak heat shock element 1,464 bp upstream of the transcription initiation site, an atypical TATA box, and further consensus sequences typical for eukaryotic promoters such as an SP-1 binding site. Southern blot analysis indicates that PPK exists as a single-copy gene in the porcine haploid genome. The PPK gene is transcribed in all investigated tissues as shown by Northern blotting and reverse transcriptase polymerase chain reaction. Comparison of the protein and cDNA sequences of PPK to other sequences in DNA and protein databases indicates significant homology to a class of heat shock proteins, the glucose-regulated proteins (GRP94). In addition, nucleotide sequences at the 5' terminus of the PPK gene show strong homology to the GRP94 family. Domains highly conserved with human tumor rejection antigen (GP96) or glucose-regulated protein (GRP94) genes are identified within the 5' terminus and the first intron of the PPK gene. These findings suggest that these proteins are either identical or represent a family of closely related proteins.

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