Platelet-derived growth factor triggers PKA-mediated signalling by a redox-dependent mechanism in rat renal mesangial cells

Inflammatory glomerular kidney diseases are often accompanied with a massive production of reactive oxygen species (ROS) that affect the function of the glomerular filtration barrier and contribute to mesangiolysis via the induction of cell death in mesangial cells. Intriguingly, ROS also trigger fine-tuned signalling processes that affect gene expression and cell proliferation or migration. To define such redox-driven signalling devices, a proteomics approach was performed to identify the formation of protein complexes induced by ROS. To this end, protein lysates of human podocytes were treated with or without hydrogen peroxide (250 μM). Thereafter cell lysates were subjected to diagonal 2D gel electrophoresis and putative redox-affected proteins were analysed by MS/MS analysis. Among others, the regulatory subunit of protein kinase A (PKA) could be identified that forms homodimers under oxidative conditions. To evaluate whether ROS dependent dimerization of PKA also occurs in a more physiological setting, rat mesangial cells were treated with platelet-derived growth factor-BB (PDGF-BB) to induce ROS formation. This regimen resulted in a redox dependent dimerization of the R-subunits of PKA. To demonstrate whether PDGF-BB induced ROS formation affects PKA dependent pathways, the effects of PDGF-BB on phosphorylation of serine 157 of vasodilator stimulated protein (VASP) a classical target of PKA were analysed. Interestingly PDGF-BB induced VASP phosphorylation in a ROS dependent manner but independent of changes in cAMP levels. Taken together, we demonstrate a redox-mediated activation of PKA by PDGF-BB thus highlighting a physiological role of ROS as regulator of PKA activity in rat mesangial cells.     

Chemical hazards present in liquids and vapors of electronic cigarettes

Electronic (e-)cigarettes have emerged in recent years as putative alternative to conventional tobacco cigarettes. These products do not contain typical carcinogens that are present in tobacco smoke, due to the lack of combustion. However, besides nicotine, hazards can also arise from other constituents of liquids, such as solvents, flavors, additives and contaminants. In this study, we have analyzed 28 liquids of seven manufacturers purchased in Germany. We confirm the presence of a wide range of flavors to enhance palatability. Although glycerol and propylene glycol were detected in all samples, these solvents had been replaced by ethylene glycol as dominant compound in five products. Ethylene glycol is associated with markedly enhanced toxicological hazards when compared to conventionally used glycerol and propylene glycol. Additional additives, such as coumarin and acetamide, that raise concerns for human health were detected in certain samples. Ten out of 28 products had been declared “free-of-nicotine” by the manufacturer. Among these ten, seven liquids were identified containing nicotine in the range of 0.1–15 µg/ml. This suggests that “carry over” of ingredients may occur during the production of cartridges. We have further analyzed the formation of carbonylic compounds in one widely distributed nicotine-free brand. Significant amounts of formaldehyde, acetaldehyde and propionaldehyde were only found at 150 °C by headspace GC–MS analysis. In addition, an enhanced formation of aldehydes was found in defined puff fractions, using an adopted machine smoking protocol. However, this effect was delayed and only observed during the last third of the smoking procedure. In the emissions of these fractions, which represent up to 40 % of total vapor volume, similar levels of formaldehyde were detected when compared to conventional tobacco cigarettes. By contrast, carbonylic compounds were hardly detectable in earlier collected fractions. Our data demonstrate the necessity of standardized machine smoking protocols to reliably address putative risks of e-cigarettes for consumers.     

Human C3 mutation reveals a mechanism of dense deposit disease pathogenesis and provides insights into complement activation and regulation

Dense deposit disease (DDD) is a severe renal disease characterized by accumulation of electron-dense material in the mesangium and glomerular basement membrane. Previously, DDD has been associated with deficiency of factor H (fH), a plasma regulator of the alternative pathway (AP) of complement activation, and studies in animal models have linked pathogenesis to the massive complement factor 3 (C3) activation caused by this deficiency. Here, we identified a unique DDD pedigree that associates disease with a mutation in the C3 gene. Mutant C3_923ΔDG, which lacks 2 amino acids, could not be cleaved to C3b by the AP C3-convertase and was therefore the predominant circulating C3 protein in the patients. However, upon activation to C3b by proteases, or to C3(H₂O) by spontaneous thioester hydrolysis, C3_923ΔDG generated an active AP C3-convertase that was regulated normally by decay accelerating factor (DAF) but was resistant to decay by fH. Moreover, activated C3b_923ΔDG and C3(H₂O)_923ΔDG were resistant to proteolysis by factor I (fI) in the presence of fH, but were efficiently inactivated in the presence of membrane cofactor protein (MCP). These characteristics cause a fluid phase–restricted AP dysregulation in the patients that continuously activated and consumed C3 produced by the normal C3 allele. These findings expose structural requirements in C3 that are critical for recognition of the substrate C3 by the AP C3-convertase and for the regulatory activities of fH, DAF, and MCP, all of which have implications for therapeutic developments.     

Recurrent deletion of 3p13 targets multiple tumour suppressor genes and defines a distinct subgroup of aggressive ERG fusion‐positive prostate cancers

Deletion of 3p13 has been reported from about 20% of prostate cancers. The clinical significance of this alteration and the tumour suppressor gene(s) driving the deletion remain to be identified. We have mapped the 3p13 deletion locus using SNP array analysis and performed fluorescence in situ hybridization (FISH) analysis to search for associations between 3p13 deletion, prostate cancer phenotype and patient prognosis in a tissue microarray containing more than 3200 prostate cancers. SNP array analysis of 72 prostate cancers revealed a small deletion at 3p13 in 14 (19%) of the tumours, including the putative tumour suppressors FOXP1, RYBP and SHQ1. FISH analysis using FOXP1‐specific probes revealed deletions in 16.5% and translocations in 1.2% of 1828 interpretable cancers. 3p13 deletions were linked to adverse features of prostate cancer, including advanced stage (p < 0.0001), high Gleason grade (p = 0.0125), and early PSA recurrence (p = 0.0015). In addition, 3p13 deletions were linked to ERG⁺ cancers and to PTEN deletions (p < 0.0001 each). A subset analysis of ERG⁺ tumours revealed that 3p13 deletions occurred independently from PTEN deletions (p = 0.3126), identifying tumours with 3p13 deletion as a distinct molecular subset of ERG⁺ cancers. mRNA expression analysis confirmed that all 3p13 genes were down regulated by the deletion. Ectopic over‐expression of FOXP1, RYBP and SHQ1 resulted in decreased colony‐formation capabilities, corroborating a tumour suppressor function for all three genes. In summary, our data show that deletion of 3p13 defines a distinct and aggressive molecular subset of ERG⁺ prostate cancers, which is possibly driven by inactivation of multiple tumour suppressors. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Transient enriched housing before amyloidosis onset sustains cognitive improvement in Tg2576 mice

Levels of educational and occupational attainment, as components of cognitive reserve, may modify the relationship between the pathological hallmarks and cognition in Alzheimer's disease (AD). We examined whether exposure of a Tg2576 transgenic mouse model of AD to environmental enrichment (EE) at a specific period during the amyloidogenic process favored the establishment of a cognitive reserve. We found that exposure to EE during early adulthood of Tg2576 mice—before amyloidogenesis has started—reduced the severity of AD-related cognitive deficits more efficiently than exposure later in life, when the pathology is already present. Interestingly, early-life exposure to EE, while slightly reducing forebrain surface covered by amyloid plaques, did not significantly impact aberrant inhibitory remodeling in the hippocampus of Tg2576 mice. Thus, transient early-life exposure to EE exerts long-lasting protection against cognitive impairment during AD pathology. In addition, these data define the existence of a specific life time frame during which stimulatory activity most efficiently builds a cognitive reserve, limiting AD progression and favoring successful aging.