Involvement of MAPKs in endostatin-mediated regulation of blood-retinal barrier function

Purpose: This study aimed to evaluate the effects of endostatin on tight junction (TJ) integrity in retinal microvascular endothelial cells (RMECs) in vitro and in vivo. Moreover, it was hypothesized that endostatin-induced occludin upregulation regulated VEGF(165)-mediated increases in endothelial cell permeability and involved activation of the MAPK signaling cascade. Endostatin is a 20-kDa fragment of collagen XVIII that has been shown to be efficacious in the eye by preventing retinal neovascularization. Endostatin is a specific inhibitor of endothelial cell proliferation, migration, and angiogenesis and has been reported to reverse VEGF-mediated increases in vasopermeability and to promote integrity of the blood-retinal barrier (BRB). In order to determine the mechanism of endostatin action on BRB integrity, we have examined the effects of endostatin on a number of intracellular pathways implicated in endothelial cell physiology. Methods: C57/Bl6 mice were injected with VEGF(165) and/or endostatin, and the distribution of occludin staining was determined using retinal flatmounts. Western blot analysis of RMECs treated with VEGF(165) and/or endostatin was used to determine changes in occludin expression and p38 MAPK and extracellular regulated kinase (ERK1/ERK2 MAPK) activation, while FD-4 flux across the RMEC monolayer was used to determine changes in paracellular permeability. Results: Endostatin prevented the discontinuous pattern of occludin staining observed at the retinal blood vessels of mice administered an intraocular injection of VEGF(165). It was shown that endostatin activated p38 MAPK 5 min after addition to RMECs and continued to do so for approximately 30 min. Endostatin was also shown to activate ERK1/ERK2 5 min after addition and continued to do so, albeit with less potency, up to and including 15 min after addition. Inhibition of p38 MAPK and ERK1/ERK2 prevented endostatin's ability to upregulate levels of occludin expression. Inhibition of these key signaling molecules was shown to prevent endostatin's ability to protect against VEGF(165)- mediated increases in paracellular permeability in vitro. However, it appears that p38 MAPK may play a more important role in VEGF-mediated permeability, as inhibition of ERK1/ERK2 will not prevent VEGF(165)- mediated permeability compared with control ( untreated) cells or cells treated with both a p38 MAPK inhibitor and VEGF(165). Conclusions: Occludin is important for the maintenance of tight junction integrity in vivo. In a p38 MAPK and ERK1/ERK2 dependent manner, endostatin was shown to upregulate the levels of expression of the tight junction protein occludin. Inhibition of these key MAPK components may prevent endostatin's ability to decrease VEGF(165)-induced paracellular permeability.

Global down-regulation of HOX gene expression in PML-RAR alpha(+) acute promyelocytic leukemia identified by small-array real-time PCR

Acute promyelocytic leukemia (APL) is associated with a reciprocal and balanced translocation involving the retinoic acid receptor-alpha (RARalpha). All-trans retinoic acid (ATRA) is used to treat APL and is a potent morphogen that regulates HOX gene expression in embryogenesis and organogenesis. HOX genes are also involved in hematopoiesis and leukemogenesis. Thirty-nine mammalian HOX genes have been identified and classified into 13 paralogous groups clustered on 4 chromosomes. They encode a complex net-Work of transcription regulatory proteins whose precise targets remain poorly understood. The overall function of the network appears to be dictated by gene dosage. To investigate the mechanisms involved in HOX gene regulation in hematopoiesis and leukemogenesis by precise measurement of individual HOX genes, a small-array real-time HOX (SMART-HOX) quantitative polymerase chain reaction (PCR) platform was designed and validated. Application of SMART-HOX to 16 APL bone marrow samples revealed a global down-regulation of 26 HOX genes compared with normal controls. HOX gene expression was also altered during differentiation induced by ATRA in the PML-RARalpha(+) NB4 cell line. PML-RARalpha, fusion proteins have been reported to act as part of a repressor complex during myelold cell differentiation, and a model linking HOX gene expression to this PML-RARalpha repressor complex is now proposed.

Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae

Tigecycline resistance has been attributed to ramA overexpression and subsequent acrA upregulation. The ramA locus, originally identified in Klebsiella pneumoniae, has homologues in Enterobacter and Salmonella spp. In this study, we identify in silico that the ramR binding site is also present in Citrobacter spp. and that Enterobacter, Citrobacter and Klebsiella spp. share key regulatory elements in the control of the romA–ramA locus. RACE (rapid amplification of cDNA ends) mapping indicated that there are two promoters from which romA–ramA expression can be regulated in K. pneumoniae. Correspondingly, electrophoretic binding studies clearly showed that purified RamA and RamR proteins bind to both of these promoters. Hence, there appear to be two RamR binding sites within the Klebsiella romA–ramA locus. Like MarA, RamA binds the promoter region, implying that it might be subject to autoregulation. We have identified changes within ramR in geographically distinct clinical isolates of K. pneumoniae. Intriguingly, levels of romA and ramA expression were not uniformly affected by changes within the ramR gene, thereby supporting the dual promoter finding. Furthermore, a subset of strains sustained no changes within the ramR gene but which still overexpressed the romA–ramA genes, strongly suggesting that a secondary regulator may control ramA expression.

Protein kinase B/Akt regulation in diabetic kidney disease

Many reviews have been written on protein kinase B/Akt focusing on its history dating back from the isolation of the Akt8 transforming murine leukemia virus by Staal in 1977, to the co-discovery of the Akt1 gene by the three groups in 1991 (reviewed in 7). There are currently over 22,000 publications in the PubMed database with "Akt" as a keyword - these publications describe a wealth of diverse data on the physiological functions of Akt isoforms. Many of these publications describe roles of Akt ranging from its requirement for cellular processes such as glucose uptake, cell survival and angiogenesis to roles in diseases such as cancer and ischaemia (22). This review will focus on the evidence for Akt signaling in different kidney cells during diabetes, or diabetic nephropathy (DN).

Extracellular BMP-antagonist regulation in development and disease: tied up in knots

Developmental processes are regulated by the bone morphogenetic protein (BMP) family of secreted molecules. BMPs bind to serine/threonine kinase receptors and signal through the canonical Smad pathway and other intracellular effectors. Integral to the control of BMPs is a diverse group of secreted BMP antagonists that bind to BMPs and prevent engagement with their cognate receptors. Tight temporospatial regulation of both BMP and BMP-antagonist expression provides an exquisite control system for developing tissues. Additional facets of BMP-antagonist biology, such as crosstalk with Wnt and Sonic hedgehog signaling during development, have been revealed in recent years. In addition, previously unappreciated roles for the BMP antagonists in kidney fibrosis and cancer have been elucidated. This review provides a description of BMP-antagonist biology, together with highlights of recent novel insights into the role of these antagonists in development, signal transduction and human disease.

Association of LETM1 and MRPL36 Contributes to the Regulation of Mitochondrial ATP Production and Necrotic Cell Death

Leucine zipper/EF hand-containing transmembrane-1 (LETM1) is a mitochondrial inner membrane protein that was first identified in Wolf-Hirschhorn syndrome, and was deleted in nearly all patients with the syndrome. LETM1 encodes for the human homologue of yeast Mdm38p, which is a mitochondria-shaping protein of unclear function. Here, we describe LETM1-mediated regulation of mitochondrial ATP production and biogenesis. We show that LETM1 overexpression can induce necrotic cell death in HeLa cells, in which LETM1 reduces mitochondria) biogenesis and ATP production. LETM1 acts as an anchor protein and associates with mitochondrial ribosome protein L36. Adenovirus-mediated overexpression of LETM1 reduced mitochondrial mass and expression of many mitochondrial proteins. LETM1-mediated inhibition of mitochondrial biogenesis enhanced glycolytic ATP supply and activated protein kinase B activity and cell survival signaling. The expression levels of LETM1 were significantly increased in multiple human cancer tissues compared with normals. These data suggest that LETM1 serves as an anchor protein for complex formation with the mitochondrial ribosome and regulates mitochondrial biogenesis. The increased expression of LETM1 in human cancer suggests that deregulation of LETM1 is a key feature of tumorigenesis. [Cancer Res 2009;69(8):3397-404]

Regulation of OPA1-mediated mitochondrial fusion by leucine zipper/EF-hand-containing transmembrane protein-1 plays a role in apoptosis

Carboxyl-terminal modulator protein (CTMP) is a tumor suppressor-like binding partner of Protein kinase B (PKB/Akt) that negative regulates this kinase. In the course of our recent work, we identified that CTMP is consistently associated with leucine zipper/EF-hand-containing transmembrane-1 (LETM1). Here, we report that adenovirus-LETM1 increased the sensitivity of HeLa cells to apoptosis, induced by either staurosporine or actinomycin D. As shown previously, LETM1 localized to the inner mitochondrial membrane. Electron-microscopy analysis of adenovirus-LETM1 transduced cells revealed that mitochondrial cristae were swollen in these cells, a phenotype similar to that observed in optic atrophy type-1 (OPA1)-ablated cells. OPA1 cleavage was increased in LETM1-overexpressing cells, and this phenotype was reversed by overexpression of OPA1 variant-7, a cleavage resistant form of OPA1. Taken together, these data suggest that LETM1 is a novel binding partner for CTMP that may play an important role in mitochondrial fragmentation via OPA1-cleavage. (C) 2009 Elsevier Inc. All rights reserved

p63 maintains keratinocyte proliferative capacity through regulation of Skp2-p130 levels

p63 is a master regulator of proliferation and differentiation in stratifying epithelia, and its expression is frequently altered in carcinogenesis. However, its role in maintaining proliferative capacity remains unclear. Here, we demonstrate that hypoproliferation and loss of differentiation in organotypic raft cultures of primary neonatal human foreskin keratinocytes (HFKs) depleted of the a and ß isoforms of p63 result from p53-p21-mediated accumulation of retinoblastoma (Rb) family member p130. Hypoproliferation in p63-depleted HFKs can be rescued by depletion of p53, p21(CIP1) or p130. Furthermore, we identified the gene encoding S-phase kinase-associated protein 2 (Skp2), the recognition component of the SCF(Skp2) E3 ubiquitin ligase, as a novel target of p63, potentially influencing p130 levels. Expression of Skp2 is maintained by p63 binding to a site in intron 2 and mRNA levels are downregulated in p63-depleted cells. Hypoproliferation in p63-depleted cells can be restored by re-expression of Skp2. Taken together, these results indicate that p63 plays a multifaceted role in maintaining proliferation in the mature regenerating epidermis, in addition to being required for differentiation.

A20 Regulation of NF-{kappa}B: Perspectives for Inflammatory Lung Disease.

Persistent activation of NF-B is central to the pathogenesis of many inflammatory lung disorders including Cystic Fibrosis, Asthma and Chronic Obstructive Pulmonary Disease. A20 is an endogenous negative regulator of NF-B signalling which has been widely described in autoimmune and inflammatory disorders including Diabetes and Crohn’s disease, but which has received little attention in terms of chronic lung disorders. This review examines the existing body of research on A20 regulation of NF-B signalling and details the mechanism and regulation of A20 action focusing, where possible, on pulmonary inflammation. A20 and its associated signalling molecules are highlighted as being of potential therapeutic interest for the treatment of inflammatory disorders and a proposed model of A20 activity in inflammatory lung disease is provided.

Comprehensive genomic screens identify a role for PLZF-RAR alpha as a positive regulator of cell proliferation via direct regulation of c-MYC

The t(11; 17)(q23;q21) translocation is associated with a retinoic acid (RA)-insensitive form of acute promyelocytic leukemia (APL), involving the production of reciprocal fusion proteins, promyelocytic leukemia zinc finger-retinoic acid receptor alpha (PLZF-RAR alpha) and RAR alpha-PLZF. Using a combination of chromatin immuno-precipitation promotor arrays (ChIP-chip) and gene expression profiling, we identify novel, direct target genes of PLZF-RAR alpha that tend to be repressed in APL compared with other myeloid leukemias, supporting the role of PLZF-RAR alpha as an aberrant repressor in APL. In primary murine hematopoietic progenitors, PLZF-RAR alpha promotes cell growth, and represses Dusp6 and Cdkn2d, while inducing c-Myc expression, consistent with its role in leukemogenesis. PLZF-RAR alpha binds to a region of the c-MYC promoter overlapping a functional PLZF site and antagonizes PLZF-mediated repression, suggesting that PLZF-RAR alpha may act as a dominant-negative version of PLZF by affecting the regulation of shared targets. RA induced the differentiation of PLZF-RAR alpha-transformed murine hematopoietic cells and reduced the frequency of clonogenic progenitors, concomitant with c-Myc down-regulation. Surviving RA-treated cells retained the ability to be replated and this was associated with sustained c-Myc expression and repression of Dusp6, suggesting a role for these genes in maintaining a self-renewal pathway triggered by PLZF-RAR alpha. (Blood. 2009; 114: 5499-5511)

Regulation of cyclin D1 by the BRCA1-BARD1 complex

Background BRCA1 and cyclin D₁ are both essential for normal breast development and mutation or aberration of their expression is associated with breast cancer [1,2]. Cyclin D₁ is best known as a G₁ cyclin where it regulates the G₁ to S phase transition by acting as a rate-limiting subunit of CDK4/6 kinase activity. More recently, however, Stacey has demonstrated that cyclin D₁ levels in G₂/M determine whether a cell continues to proliferate or exits the cell cycle [3]. The majority of BRCA1 in the cell is bound to BARD1 through their N-terminal RING domains. Heterodimerization is essential for the stability and correct localization of the complex and confers ubiquitin ligase activity to BRCA1. The importance of the ligase activity of BRCA1 to breast cancer development is inferred from the fact that N-terminal diseaseassociated mutations are proposed to reduce ligase activity [4]. Methods Protein–protein interactions were demonstrated using yeast-two-hybrid and coimmunoprecipitation. Protein levels were altered through overexpression, siRNA and antisense technology. The effect of proteasome inhibitors and cycloheximide treatment was also examined. Results We initially identified cyclin D₁ as a binding partner of BARD1 in a yeast-two-hybrid screen and defined the minimal binding region as the N-terminus of BARD1. This interaction was confirmed in vivo by coimmunoprecipitation. The N-terminus of BARD1 also binds BRCA1 and imparts ubiquitin ligase activity to the complex. Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Traditionally polyubiquitin chains linked through lysine 48 target proteins for degradation by the 26 S proteasome. We have demonstrated that cyclin D₁ protein levels are inversely related to BRCA1 and BARD1 levels in several model systems. Furthermore, regulation of cyclin D1 levels occurs through a post-transcriptional mechanism and requires the ligase activity of BRCA1. Interestingly, this phenomenon is cell-cycle regulated, occurring in G₂/M. Conclusion We propose that cyclin D₁ is a potential substrate for BRCA1 ubiquitination and that this targets cyclin D₁ for proteasomal-mediated degradation. Future work will focus on ascertaining the functional consequence of cyclin D₁ regulation by the BRCA1–BARD1 complex; in particular, the impact of BRCA1, mediated through regulation of cyclin D₁, on the proliferation versus differentiation decision.

MicroRNA regulation of core apoptosis pathways in cancer

Recent research has demonstrated that microRNAs (miRNAs) are key regulators of many cell processes often deregulated in cancer, including apoptosis. Indeed, it is becoming clear that many miRNAs are anti-apoptotic and mediate this effect by targeting pro-apoptotic mRNAs or positive regulators of pro-apoptotic mRNAs. Conversely, many pro-apoptotic miRNAs target anti-apoptotic mRNAs or their positive regulators. We have reviewed the current knowledge in this area including evidence of miRNA involvement in cancer drug resistance. (C) 2010 Elsevier Ltd. All rights reserved.

Young People, Conflict and Regulation

Set against the progress claimed since the Good Friday/ Belfast Agreement, this article reflects the reality of life for children and young people as they negotiate the aftermath of the Conflict in Northern Ireland. Their experiences of informal and formal policing, community and State control, demonstrate the need to understand the lasting impacts of the Conflict when developing policies and practices affecting their lives. At a crucial defining period in the devolution of justice and policing, and based on primary research conducted by the authors, the article establishes key rights-compliant principles central to reform of youth justice.