Co-regulation of Gremlin and Notch signalling in diabetic nephropathy

Diabetic nephropathy is currently the leading cause of end-stage renal disease worldwide, and occurs in approximately one third of all diabetic patients. The molecular pathogenesis of diabetic nephropathy has not been fully characterized and novel mediators and drivers of the disease are still being described. Previous data from our laboratory has identified the developmentally regulated gene Gremlin as a novel target implicated in diabetic nephropathy in vitro and in vivo. We used bioinformatic analysis to examine whether Gremlin gene sequence and structure could be used to identify other genes implicated in diabetic nephropathy. The Notch ligand Jagged1 and its downstream effector, hairy enhancer of split-1 (Hes1), were identified as genes with significant similarity to Gremlin in terms of promoter structure and predicted microRNA binding elements. This led us to discover that transforming growth factor-beta (TGFß1), a primary driver of cellular changes in the kidney during nephropathy, increased Gremlin, Jagged1 and Hes1 expression in human kidney epithelial cells. Elevated levels of Gremlin, Jagged1 and Hes1 were also detected in extracts from renal biopsies from diabetic nephropathy patients, but not in control living donors. In situ hybridization identified specific upregulation and co-expression of Gremlin, Jagged1 and Hes1 in the same tubuli of kidneys from diabetic nephropathy patients, but not controls. Finally, Notch pathway gene clustering showed that samples from diabetic nephropathy patients grouped together, distinct from both control living donors and patients with minimal change disease. Together, these data suggest that Notch pathway gene expression is elevated in diabetic nephropathy, co-incident with Gremlin, and may contribute to the pathogenesis of this disease.

Advances in protein kinase B signalling: AKTion on multiple fronts

The role of the serine/threonine protein kinase B (PKB, also known as Akt) is becoming increasingly more evident to researchers investigating diverse cellular processes such as glucose uptake, cell-cycle progression, apoptosis and transcriptional regulation. New roles for PKB/Akt have been described in various organisms and biological processes. From the regulation of ovarian ecdysteroid production in the humble mosquito (Aedes aegypti), through the seasonal, tissue-specific regulation of PKB/Akt during the hibernation of yellow-bellied marmots (Marmota flaviventris), to the control of glucose metabolism and insulin signalling in the mouse (Mus musculus), our knowledge of the function of this protein kinase has expanded greatly in recent years. Significant advances in all aspects of PKB/Akt signalling have occurred in the past 2 years, including biological insights, novel substrates and newly discovered regulatory mechanisms of PKB/Akt. Collectively, these data expand the current models of PKB/Akt signalling and highlight potential directions for PKB/Akt research in the future.

Ten years of protein kinase B signalling: a hard Akt to follow

It is ten years since the publication of three papers describing the cloning of a new proto-oncogene serine/threonine kinase termed protein kinase B (PKB)/Akt. Key roles for this protein kinase in cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription and cell migration are now well established. The explosion of publications involving PKB/Akt in the past three years emphasizes the high level of current interest in this signalling molecule. This review focuses on tracing the characterization of this kinase, through the elucidation of its mechanism of regulation, to its role in regulating physiological and pathophysiological processes,to our current understanding of the biology of PKB/Akt, and prospects for the future.

Neuropeptide signalling systems in flatworms

Two distinct families of neuropeptides are known to endow platyhelminth nervous systems-the FMRFamide-like peptides (FLPs) and the neuropepticle Fs (NPFs). Flatworm FLPs are strusturally simple, each 4-6 amino acids in length with a carboxy terminal aromatic-hydropliobic-Arg-Phe-amide motif. Thus far, four distinct flatworm FLPs have been characterized, with only one of these from a parasite. They have a widespread distribution within the central and peripheral nervous system of every flatworm examined, including neurones serving the attachment organs, the somatic Musculature and the reproductive system. The only physiological role that has been identified for flatworm FLPs is myoexcitation. Flatworm NPFs are believed to be invertebrate homologues of the vertebrate neuropeptide Y (NPY) family of peptides. Flatworm NPFs are 36-39 amino acids in length and are characterized by a caboxy terminal GRPRFarnide signature and conserved tyrosine residues at positions 10 and 17 from the carboxy terminal. Like FLPs, NPF occurs throughout flatworm nervous systems, although less is known about its biological role. While there is some evidence for a myoexcitatory action in cestodes and flukes, more compelling physiological data indicate that flatworm NPF inhibits cAMP levels in a manner that is characteristic of NPY action in vertebrates. The widespread expression of these neuropeptides in flanworm parasites highlights the potential of these signalling systems to yield new targets for novel anthelmintics. Although platyhelminth FLP and NPF receptors await identification, other molecules that play pivotal roles in neuropeptide signalling have been uncovered. These enzymes, involved in the biosynthesis and processing of flatworm neuropeptides, have recently been described and offer other distinct and attractive targets for therapeutic interference.

New molecular targets in radiotherapy: DNA damage signalling and repair in targeted and non-targeted cells

Ionising radiation plays a key role in therapy due to its ability to directly induce DNA damage, in particular DNA double-strand breaks leading to cell death. Cells have multiple repair pathways which attempt to maintain genomic stability. DNA repair proteins have become key targets for therapy, using small molecule inhibitors, in combination with radiation and or chemotherapeutic agents as a means of enhancing cell killing. Significant advances in our understanding of the response of cells to radiation exposures has come from the observation of non-targeted effects where cells respond via mechanisms other than those which are a direct consequence of energy-dependent DNA damage. Typical of these is bystander signalling where cells respond to the fact that their neighbours have been irradiated. Bystander cells show a DNA damage response which is distinct from directly irradiated cells. In bystander cells, ATM- and Rad3-related (ATR) protein kinase-dependent signalling in response to stalled replication forks is an early event in the DNA damage response. The ATM protein kinase is activated downstream of ATR in bystander cells. This offers the potential for differential approaches for the modulation of bystander and direct effects with repair inhibitors which may impact on the response of tumours and on the protection of normal tissues during radiotherapy. (C) 2009 Elsevier B.V. All rights reserved.

VEGF-induced retinal angiogenic signalling is critically dependent on Ca2+ signalling via Ca2+/calmodulin-dependent protein kinase II

Purpose. The authors conducted an in vitro investigation of the role of Ca2+-dependent signaling in vascular endothelial growth factor (VEGF)-induced angiogenesis in the retina.

Methods. Bovine retinal endothelial cells (BRECs) were stimulated with VEGF in the presence or absence of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM; intracellular Ca2+ chelator), U73122 (phospholipase C (PLC) inhibitor), xestospongin C (Xe-C), and 2-aminoethoxydiphenyl borate (2APB) (inhibitors of inositol-1,4,5 triphosphate (IP3) signaling). Intracellular Ca2+ concentration ([Ca2+]i) was estimated using fura-2 Ca2+ microfluorometry, Akt phosphorylation quantified by Western blot analysis, and angiogenic responses assessed using cell migration, proliferation, tubulogenesis, and sprout formation assays. The effects of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 were also evaluated on VEGF-induced Akt signaling and angiogenic activity.

Results. Stimulation of BRECs with 25 ng/mL VEGF induced a biphasic increase in [Ca2+]i, with an initial transient peak followed by a sustained plateau phase. VEGF-induced [Ca2+]i increases were almost completely abolished by pretreating the cells with BAPTA-AM, U73122, Xe-C, or 2APB. These agents also inhibited VEGF-induced phosphorylation of Akt, cell migration, proliferation, tubulogenesis, and sprouting angiogenesis. KN93 was similarly effective at blocking the VEGF-induced activation of Akt and angiogenic responses.

Conclusions. VEGF increases [Ca2+]i in BRECs through activation of the PLC-IP3 signal transduction pathway. VEGF-induced phosphorylation of the proangiogenic protein Akt is critically dependent on this increase in [Ca2+]i and the subsequent activation of CaMKII. Pharmacologic inhibition of Ca2+-mediated signaling in retinal endothelial cells blocks VEGF-induced angiogenic responses. These results suggest that the PLC/IP3/Ca2+/CaMKII signaling pathway may be a rational target for the treatment of angiogenesis-related disorders of the eye.

Jagged/Notch signalling is required for a subset of TGFβ1 responses in human kidney epithelial cells.

The Jagged/Notch pathway has been implicated in TGFß1 responses in epithelial cells in diabetic nephropathy and other fibrotic conditions in vivo. Here, we identify that Jagged/Notch signalling is required for a subset of TGFß1-stimulated gene responses in human kidney epithelial cells in vitro. TGFß1 treatment of HK-2 and RPTEC cells for 24 h increased Jagged1 (a Notch ligand) and Hes1 (a Notch target) mRNA. This response was inhibited by co-incubation with Compound E, an inhibitor of ?-secretase (GSI), an enzyme required for Notch receptor cleavage and transcription regulation. In both cell types, TGFß1-responsive genes associated with epithelial–mesenchymal transition such as E-cadherin and vimentin were also affected by ?-secretase inhibition, but other TGFß1 targets such as connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1) were not. TGFß1-induced changes in Jagged1 expression preceded EMT-associated gene changes, and co-incubation with GSI altered TGFß1-induced changes in cell shape and cytoskeleton. Transfection of cells with the activated, cleaved form of Notch (NICD) triggered decreased expression of E-cadherin in the absence of TGFß1, but did not affect a-smooth muscle actin expression, suggesting differential requirements for Notch signalling within the TGFß1-responsive gene subset. Increased Jagged1 expression upon TGFß1 exposure required Smad3 signalling, and was also regulated by PI3K and ERK. These data suggest that Jagged/Notch signalling is required for a subset of TGFß1-responsive genes, and that complex signalling pathways are involved in the crosstalk between TGFß1 and Notch cascades in kidney epithelia.


--------------------------------------------------------------------------------