Deficient Dopamine D2 Receptor Function Causes Renal Inflammation Independently of High Blood Pressure

Renal dopamine receptors participate in the regulation of blood pressure. Genetic factors, including polymorphisms of the dopamine D2 receptor gene (DRD2) are associated with essential hypertension, but the mechanisms of their contribution are incompletely understood. Mice lacking Drd2 (D2−/−) have elevated blood pressure, increased renal expression of inflammatory factors, and renal injury. We tested the hypothesis that decreased dopamine D2 receptor (D2R) function increases vulnerability to renal inflammation independently of blood pressure, is an immediate cause of renal injury, and contributes to the subsequent development of hypertension. In D2−/− mice, treatment with apocynin normalized blood pressure and decreased oxidative stress, but did not affect the expression of inflammatory factors. In mouse RPTCs Drd2 silencing increased the expression of TNFα and MCP-1, while treatment with a D2R agonist abolished the angiotensin II-induced increase in TNF-α and MCP-1. In uni-nephrectomized wild-type mice, selective Drd2 silencing by subcapsular infusion of Drd2 siRNA into the remaining kidney produced the same increase in renal cytokines/chemokines that occurs after Drd2 deletion, increased the expression of markers of renal injury, and increased blood pressure. Moreover, in mice with two intact kidneys, short-term Drd2 silencing in one kidney, leaving the other kidney undisturbed, induced inflammatory factors and markers of renal injury in the treated kidney without increasing blood pressure. Our results demonstrate that the impact of decreased D2R function on renal inflammation is a primary effect, not necessarily associated with enhanced oxidant activity, or blood pressure; renal damage is the cause, not the result, of hypertension. Deficient renal D2R function may be of clinical relevance since common polymorphisms of the human DRD2 gene result in decreased D2R expression and function

Phosphorylated Ribosomal Protein S6 Is Required for Akt-Driven Hyperplasia and Malignant Transformation, but Not for Hypertrophy, Aneuploidy and Hyperfunction of Pancreatic β-Cells

Constitutive expression of active Akt (Akttg) drives hyperplasia and hypertrophy of pancreatic β-cells, concomitantly with increased insulin secretion and improved glucose tolerance, and at a later stage the development of insulinoma. To determine which functions of Akt are mediated by ribosomal protein S6 (rpS6), an Akt effector, we generated mice that express constitutive Akt in β-cells in the background of unphosphorylatable ribosomal protein S6 (rpS6P-/-). rpS6 phosphorylation deficiency failed to block Akttg-induced hypertrophy and aneuploidy in β-cells, as well as the improved glucose homeostasis, indicating that Akt carries out these functions independently of rpS6 phosphorylation. In contrast, rpS6 phosphorylation deficiency efficiently restrained the reduction in nuclear localization of the cell cycle inhibitor p27, as well as the development of Akttg-driven hyperplasia and tumor formation in β-cells. In vitro experiments with Akttg and rpS6P-/-;Akttg fibroblasts demonstrated that rpS6 phosphorylation deficiency leads to reduced translation fidelity, which might underlie its anti-tumorigenic effect in the pancreas. However, the role of translation infidelity in tumor suppression cannot simply be inferred from this heterologous experimental model, as rpS6 phosphorylation deficiency unexpectedly elevated the resistance of Akttg fibroblasts to proteotoxic, genotoxic as well as autophagic stresses. In contrast, rpS6P-/- fibroblasts exhibited a higher sensitivity to these stresses upon constitutive expression of oncogenic Kras. The latter result provides a possible mechanistic explanation for the ability of rpS6 phosphorylation deficiency to enhance DNA damage and protect mice from Kras-induced neoplastic transformation in the exocrine pancreas. We propose that Akt1 and Kras exert their oncogenic properties through distinct mechanisms, even though both show addiction to rpS6 phosphorylation.Fil: Wittenberg, Avigail Dreazen. The Hebrew University Of Jerusalem; IsraelFil: Azar, Shahar. The Hebrew University Of Jerusalem; IsraelFil: Klochendler, Agnes. The Hebrew University Of Jerusalem; IsraelFil: Stolovich-Rain, Miri. The Hebrew University Of Jerusalem; IsraelFil: Avraham, Shlomit. The Hebrew University Of Jerusalem; IsraelFil: Birnbaum, Lea. The Hebrew University Of Jerusalem; IsraelFil: Binder Gallimidi, Adi. The Hebrew University Of Jerusalem; IsraelFil: Katz, Maximiliano Javier. The Hebrew University Of Jerusalem; Israel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Dor, Yuval. The Hebrew University Of Jerusalem; IsraelFil: Meyuhas, Oded. The Hebrew University Of Jerusalem; Israe

List of proteins that selectively interact with unphosphorylatable form of rpS6.

<p>Whole cell extract from HEK293 cells infected with pS6^[5S]-GFP, pS6^[5A]-GFP, pS6^[5D]-GFP or pEGFP-N1, was subjected to GFP pull-down, and the bound proteins were size fractionated by SDS-polyacrlamide gel electrophoresis. Mass spectrometric analysis of proteins in each lane was performed as described in “material and Methods” and proteins, selectively bound to pS6^[5A]-GFP in two independent experiments, are presented (numbers separated by slash [/] represent results obtained in each of the two individual analyses).</p

Small genome of the fungus Escovopsis weberi, a specialized disease agent of ant agriculture

Many microorganisms with specialized lifestyles have reduced genomes. This is best understood in beneficial bacterial symbioses, where partner fidelity facilitates loss of genes necessary for living independently. Specialized microbial pathogens may also exhibit gene loss relative to generalists. Here, we demonstrate that Escovopsis weberi, a fungal parasite of the crops of fungus-growing ants, has a reduced genome in terms of both size and gene content relative to closely related but less specialized fungi. Although primary metabolism genes have been retained, the E. weberi genome is depleted in carbohydrate active enzymes, which is consistent with reliance on a host with these functions. E. weberi has also lost genes considered necessary for sexual reproduction. Contrasting these losses, the genome encodes unique secondary metabolite biosynthesis clusters, some of which include genes that exhibit up-regulated expression during host attack. Thus, the specialized nature of the interaction between Escovopsis and ant agriculture is reflected in the parasite's genome

Constitutive expression of Akt induces polyploidy regardless of the rpS6 phosphorylation status.

<p>A. rpS6 deficiency does not affect ploidy in β-cells. Islets were isolated from wild-type (WT) and 2 month-old Akt^tg mice. Dissociated islets were fixed, permeabilized, immunostained for Insulin, HA, Ki67 and Hoechst before FACS analysis. The DNA content of 5,000 to 10,000 quiescent β-cells (insulin-positive, Ki67-negative) was measured by FACS analysis. B. 5,000 quiescent (Ki67-negative) β-cells (insulin-positive) that express myr-Akt (HA-positive) are polyploidy, whereas their HA-negative counterpart shows mostly a diploid profile as wild-type β-cells. C. rpS6 deficiency does not affect the ploidy profile of myr-Akt expressing β-cells. Islets were isolated from 2 month-old mice (three wild-type (WT), two Akt^tg and three rpS6^P-/-;Akt^tg mice) and 5,000 to 10,000 HA-positive β-cells processed as in (A) for FACS analysis.</p

rpS6 phosphorylation deficiency renders Ras^G12D MEFs more sensitive to both genotoxic and proteotoxic stresses.

<p>(A) Immortalized WT, rpS6^P-/-, Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (B) Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were seeded in 96-well plates at a density of 4 x10³ per well. Proliferation was monitored by measuring the A₆₅₀ of the methylene-blue dye extracted from stained cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149995#pone.0149995.ref032" target="_blank">32</a>]. Absorbance measured 24 h after platting, was set arbitrarily at 1 and absorbance measured at later time points (average ± SEM [n = 6]) for each time point) was normalized to that value. t_d, population-doubling time. (C) Immortalized Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were incubated with the indicated concentrations of etoposide for 24 h, harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (D) Immortalized Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were incubated with the indicated concentrations of MG132 for 24 h, harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies.</p

rpS6 phosphorylation deficiency increases proliferation and decreases translational fidelity in fibroblasts.

<p>(A) Immortalized WT, rpS6^P-/-, Akt^tg and rpS6^P-/-;Akt^tg MEFs were harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (B) MEFs described in (A) were seeded in 96-well plates at a density of 4 x10³ per well. The proliferation of MEFs described in (A) was monitored as described in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149995#pone.0149995.g004" target="_blank">Fig 4B</a>). t_d, population doubling time. (C) WT and rpS6^P-/- immortalized MEFs were incubated for 1 h with 1 mM puromycin, harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (D) Quantification of signals obtained in 6 experiments similar to that described in (C). (E) WT, rpS6^P-/-, Akt^tg, and rpS6^P-/-;Akt^tg MEFs were cotransfected with Renilla luciferase expression vector and any one of the following firefly luciferase expression vectors: Fluc(WT), Fluc(Stop) and Fluc(R218S). The transfection efficiency of any of the Fluc constructs was normalized to that of the Renilla luciferase. The relative Fluc activity of each mutant was normalized to Fluc (WT). The result obtained for each mutant in WT MEFs (n = 24) was arbitrarily set at 1 and the relative luciferase activity obtained for each Fluc mutant in rpS6^P-/- MEFs (n = 24) was normalized to that value. Similarly, results obtained for each mutant in Akt^tg MEFs (n = 12) were arbitrarily set at 1 and the relative luciferase activity obtained for each Fluc mutant in rpS6^P-/-;Akt^tg MEFs (n = 12) was normalized to that value.</p