BBA - Molecular Basis of Disease (v.1782, #1)

Acknowledgement (I-III).

Protein kinase X (PRKX) can rescue the effects of polycystic kidney disease-1 gene (PKD1) deficiency by Xiaohong Li; Christopher R. Burrow; Katalin Polgar; Deborah P. Hyink; G. Luca Gusella; Patricia D. Wilson (1-9).
Autosomal dominant polycystic kidney disease (ADPKD) is a common, genetically determined developmental disorder of the kidney that is characterized by cystic expansion of renal tubules and is caused by truncating mutations and haplo-insufficiency of the PKD1 gene. Several defects in cAMP-mediated proliferation and ion secretion have been detected in ADPKD cyst-lining epithelia. Unlike the ubiquitous PKA, the cAMP-dependent CREB-kinase, Protein Kinase X (PRKX) is developmentally regulated, tissue restricted and induces renal epithelial cell migration, and tubulogenesis in vitro as well as branching morphogenesis of ureteric bud in developing kidneys. The possibility of functional interactions between PKD1-encoded polycystin-1 and PRKX was suggested by the renal co-distribution of PRKX and polycystin-1 and the binding and phosphorylation of the C-terminal of polycystin-1 by PRKX at S4166 in vitro. Early consequences of PKD1 mutation include increased tubule epithelial cell–matrix adhesion, decreased migration, reduced ureteric bud branching and aberrant renal tubule dilation. To determine whether PRKX might counteract the adverse effects of PKD1 mutation, human ADPKD epithelial cell lines were transfected with constitutively active PRKX and shown to rescue characteristic adhesion and migration defects. In addition, the co-injection of constitutively active PRKX with inhibitory pMyr-EGFP-PKD1 into the ureteric buds of mouse embryonic kidneys in organ culture resulted in restoration of normal branching morphogenesis without cystic tubular dilations. These results suggest that PRKX can restore normal function to PKD1-deficient kidneys and have implications for the development of preventative therapy for ADPKD.
Keywords: Cyclic AMP; Renal cystic disease; Developmental biology; Adhesion; Migration; Differentiation; Morphogenesis;

Co-regulation of Gremlin and Notch signalling in diabetic nephropathy by David W. Walsh; Sarah A. Roxburgh; Paul McGettigan; Celine C. Berthier; Desmond G. Higgins; Matthias Kretzler; Clemens D. Cohen; Sergio Mezzano; Derek P. Brazil; Finian Martin (10-21).
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 (TGFβ1), 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.
Keywords: Diabetic nephropathy; Gremlin; Notch; Jagged; Co-regulation; Promoter homology;

Glycation by ascorbic acid oxidation products leads to the aggregation of lens proteins by Mikhail Linetsky; Ekaterina Shipova; Rongzhu Cheng; Beryl J. Ortwerth (22-34).
Previous studies from this laboratory have shown that there are striking similarities between the yellow chromophores, fluorophores and modified amino acids released by proteolytic digestion from calf lens proteins ascorbylated in vitro and their counterparts isolated from aged and cataractous lens proteins. The studies reported in this communication were conducted to further investigate whether ascorbic acid-mediated modification of lens proteins could lead to the formation of lens protein aggregates capable of scattering visible light, similar to the high molecular aggregates found in aged human lenses. Ascorbic acid, but not glucose, fructose, ribose or erythrulose, caused the aggregation of calf lens proteins to proteins ranging from 2.2 × 106 up to 3.0 × 10Da. This compared to proteins ranging from 1.8 × 106 up to 3.6 × 10Da for the water-soluble (WS) proteins isolated from aged human lenses. This aggregation was likely due to the glycation of lens crystallins because [U-14C] ascorbate was incorporated into the aggregate fraction and because NaCNBH3, which reduces the initial Schiff base, prevented any protein aggregation. Reactions of ascorbate with purified crystallin fractions showed little or no aggregation of α-crystallin, significant aggregation of βH-crystallin, but rapid precipitation of purified βL- and γ-crystallin. The aggregation of lens proteins can be prevented by the binding of damaged crystallins to alpha-crystallin due to its chaperone activity. Depending upon the ratios between the components of the incubation mixtures, α-crystallin prevented the precipitation of the purified βL- and γ-crystallin fractions during ascorbylation. The addition of at least 20% of alpha-crystallin by weight into glycation mixtures with βL-, or γ-crystallins completely inhibited protein precipitation, and increased the amount of the high molecular weight aggregates in solution. Static and dynamic light scattering measurements of the supernatants from the ascorbic acid-modified mixtures of α- and βL-, or γ-crystallins showed similar molar masses (up to 10Da) and hydrodynamic diameter (up to 80 nm). These data support the hypothesis, that if the lens reducing environment is compromised, the ascorbylation of lens crystallins can significantly change the short range interactions between different classes of crystallins leading to protein aggregation, light scattering and eventually to senile cataract formation.
Keywords: Ascorbic acid; Glycation; Lens proteins; Protein aggregation; Light scattering;

Tenascin C interacts with Ecto-5′-nucleotidase (eN) and regulates adenosine generation in cancer cells by Rafal Sadej; Kunihiro Inai; Zenon Rajfur; Anna Ostapkowicz; Jon Kohler; Andrzej C. Skladanowski; Beverly S. Mitchell; Jozef Spychala (35-40).
Tenascin C is expressed in invasive human solid tumors; however its specific role in cancer biology remains obscure. Previously, we have found that ecto-5′-nucleotidase (eN) is a marker of ER (−) breast carcinoma and elevated expression correlates with invasive mesenchymal cell phenotype. To investigate for the potential relationship between eN and protein components of the extracellular matrix (ECM) we measured adenosine generation from AMP in cells incubated with soluble ECM proteins. We found that tenascin C was the only ECM component that strongly inhibited ecto-5′-nucleotidase (eN) activity in situ and adenosine generation from AMP (75% inhibition, p < 0.01). The inhibition was comparable to that induced by concanavalin A, a well-defined and strong inhibitor of eN. Resin immobilized tenascin C, but not collagen, and only weakly fibronectin, specifically and quantitatively bound cell-extracted eN. We further developed breast cancer cell line with reduced eN expression and tested changes in cell adhesion on different ECM. Breast cancer cells expressing reduced eN attached 56% weaker (p < 0.05) to immobilized tenascin C. This difference was not detected with other ECM proteins. Finally, control breast cancer cells migrated slower on tenascin C when compared with clone with reduced eN expression. These data suggest that eN is a novel and specific receptor for tenascin C and that the interaction between these proteins may influence cell adhesion and migration and also lead to decreased generation of local adenosine.
Keywords: Ecto-5′-nucleotidase; CD73; Adenosine; Breast cancer; Extracellular matrix; Tenascin C; Cell adhesion; Cell motility; Invasion;

A tyrosine kinase inhibitor, β-hydroxyisovalerylshikonin, induced apoptosis in human lung cancer DMS114 cells through reduction of dUTP nucleotidohydrolase activity by Sachiko Kajimoto; Masayo Horie; Hitoshi Manabe; Yutaka Masuda; Toshiko Shibayama-Imazu; Shigeo Nakajo; Xiang Feng Gong; Takashi Obama; Hiroyuki Itabe; Kazuyasu Nakaya (41-50).
Apoptotic cell death was induced in human lung cancer DMS114 cells by treatment with β-hydroxyisovalerylshikonin (β-HIVS), an ATP-noncompetitive inhibitor of protein tyrosine kinases. Changes in phosphoprotein profiles were analyzed by two-dimensional-polyacrylamide gel electrophoresis (2D-PAGE) after the cells were treated with β-HIVS. One spot on the 2D gel showed a marked decrease in intensity and the corresponding protein was identified by mass spectrometry as dUTP nucleotidohydrolase (dUTPase). The β-HIVS-induced decrease of dUTPase in the phosphoprotein fraction of DMS114 cells was confirmed using immunoblotting. Treatment of the cells with β-HIVS-induced rapid reduction of dUTPase activity. An antioxidant N-acetyl-cysteine inhibited both the reduction of phosphorylated dUTPase and the induction of apoptosis by β-HIVS treatment of DMS114 cells. Introduction of siRNA directed against dUTPase mRNA into DMS114 cells enhanced the susceptibility of β-HIVS-induced apoptosis. Treatment of DMS114 cells with β-HIVS and 5-fluorouracil, a specific inhibitor of thymidylate synthase used as a chemotherapeutic drug, revealed the synergistic effects of these drugs on the inhibition of cell growth. These results suggest that dUTPase activity is one of the crucial factors involved in apoptotic cell death in lung cancer cells.
Keywords: Shikonin; Phosphoprotein; Apoptosis; dUTP nucleotidohydrolase; Lung cancer;

Downregulation of Par-3 expression and disruption of Par complex integrity by TGF-β during the process of epithelial to mesenchymal transition in rat proximal epithelial cells by Xiangyang Wang; Jing Nie; Qin Zhou; Wei Liu; Fengxin Zhu; Wei Chen; Haiping Mao; Ning Luo; Xiuqing Dong; Xueqing Yu (51-59).
Epithelial to mesenchymal transition (EMT) is a fundamental mechanism of organ fibrosis and the initial step is disruption of cell junction and cell polarity. TGF-β has been demonstrated as the most important mediator of EMT which is sufficient to initiate and complete the whole course of EMT, however, the detailed mechanism of TGF-β in modulating the disruption of cell junction still remains unclear. Par-3 is a component of Par complex which plays a crucial role in the establishment and maintenance of epithelial polarity. In this study, we found that TGF-β treatment resulted in a dose- and time-dependent downregulation of Par-3 protein together with the suppression of E-cadherin expression and induction of α-SMA. The decreased Par-3 subsequently resulted in the redistribution of Par-6–aPKC complex from cell membrane to cytoplasm. Forced expression of exogenous Par-3 into rat proximal epithelial cells (NRK52E) led to a drastic blockage of TGF-β1-induced E-cadherin suppression and α-SMA induction. In contrast, knockdown Par-3 expression by siRNA significantly enhanced TGF-β1-induced E-cadherin suppression and α-SMA induction. These data indicate that downregulation of Par-3 and subsequent disruption of Par complex integrity might be one mechanism that TGF-β destroys cell polarity during EMT.
Keywords: Par-3; Par complex; Cell junction; Epithelial to mesenchymal transition; TGF-β1; Proximal epithelial cell;