Kinetics and protective role of autophagy in a mouse cecal ligation and puncture-induced sepsis

INTRODUCTION: It is not well understood whether the process of autophagy is accelerated or blocked in sepsis, and whether it is beneficial or harmful to the immune defense mechanism over a time course during sepsis. Our aim was to determine both the kinetics and the role of autophagy in sepsis. METHODS: We examined autophagosome and autolysosome formation in a cecal ligation and puncture (CLP) mouse model of sepsis (in C57BL/6N mice and GFP-LC3 transgenic mice), using western blotting, immunofluorescence, and electron microscopy. We also investigated the effect of chloroquine inhibition of autophagy on these processes. RESULTS: Autophagy, as demonstrated by increased LC3-II/LC3-I ratios, is induced in the liver, heart, and spleen over 24 h after CLP. In the liver, autophagosome formation peaks at 6 h and declines by 24 h. Immunofluorescent localization of GFP-LC3 dots (alone and with lysosome-associated membrane protein type 1 (LAMP1)), as well as electron microscopic examination, demonstrate that both autophagosomes and autolysosomes are increased after CLP, suggesting that intact autophagy mechanisms operate in the liver in this model. Furthermore, inhibition of autophagy process by chloroquine administration immediately after CLP resulted in elevated serum transaminase levels and a significant increase in mortality. CONCLUSIONS: All autophagy-related processes are properly activated in the liver in a mouse model of sepsis; autophagy appears to play a protective role in septic animals

Osteocrin ameliorates adriamycin nephropathy via p38 mitogen-activated protein kinase inhibition

Natriuretic peptides exert multiple effects by binding to natriuretic peptide receptors (NPRs). Osteocrin (OSTN) binds with high affinity to NPR-C, a clearance receptor for natriuretic peptides, and inhibits degradation of natriuretic peptides and consequently enhances guanylyl cyclase-A (GC-A/NPR1) signaling. However, the roles of OSTN in the kidney have not been well clarified. Adriamycin (ADR) nephropathy in wild-type mice showed albuminuria, glomerular basement membrane changes, increased podocyte injuries, infiltration of macrophages, and p38 mitogen-activated protein kinase (MAPK) activation. All these phenotypes were improved in OSTN- transgenic (Tg) mice and NPR3 knockout (KO) mice, with no further improvement in OSTN-Tg/NPR3 KO double mutant mice, indicating that OSTN works through NPR3. On the contrary, OSTN KO mice increased urinary albumin levels, and pharmacological blockade of p38 MAPK in OSTN KO mice ameliorated ADR nephropathy. In vitro, combination treatment with ANP and OSTN, or FR167653, p38 MAPK inhibitor, reduced Ccl2 and Des mRNA expression in murine podocytes (MPC5). OSTN increased intracellular cyclic guanosine monophosphate (cGMP) in MPC5 through GC-A. We have elucidated that circulating OSTN improves ADR nephropathy by enhancing GC-A signaling and consequently suppressing p38 MAPK activation. These results suggest that OSTN could be a promising therapeutic agent for podocyte injury

Gravity sensing in plant and animal cells

Gravity determines shape of body tissue and affects the functions of life, both in plants and animals. The cellular response to gravity is an active process of mechanotransduction. Although plants and animals share some common mechanisms of gravity sensing in spite of their distant phylogenetic origin, each species has its own mechanism to sense and respond to gravity. In this review, we discuss current understanding regarding the mechanisms of cellular gravity sensing in plants and animals. Understanding gravisensing also contributes to life on Earth, e.g., understanding osteoporosis and muscle atrophy. Furthermore, in the current age of Mars exploration, understanding cellular responses to gravity will form the foundation of living in space

Findings from recent studies by the Japan Aerospace Exploration Agency examining musculoskeletal atrophy in space and on Earth

The musculoskeletal system provides the body with correct posture, support, stability, and mobility. It is composed of the bones, muscles, cartilage, tendons, ligaments, joints, and other connective tissues. Without effective countermeasures, prolonged spaceflight under microgravity results in marked muscle and bone atrophy. The molecular and physiological mechanisms of this atrophy under unloaded conditions are gradually being revealed through spaceflight experiments conducted by the Japan Aerospace Exploration Agency using a variety of model organisms, including both aquatic and terrestrial animals, and terrestrial experiments conducted under the Living in Space project of the Japan Ministry of Education, Culture, Sports, Science, and Technology. Increasing our knowledge in this field will lead not only to an understanding of how to prevent muscle and bone atrophy in humans undergoing long-term space voyages but also to an understanding of countermeasures against age-related locomotive syndrome in the elderly

DA-Raf-Mediated Suppression of the Ras—ERK Pathway Is Essential for TGF-β1-Induced Epithelial—Mesenchymal Transition in Alveolar Epithelial Type 2 Cells

<div><p>Myofibroblasts play critical roles in the development of idiopathic pulmonary fibrosis by depositing components of extracellular matrix. One source of lung myofibroblasts is thought to be alveolar epithelial type 2 cells that undergo epithelial–mesenchymal transition (EMT). Rat RLE-6TN alveolar epithelial type 2 cells treated with transforming growth factor-β1 (TGF-β1) are converted into myofibroblasts through EMT. TGF-β induces both canonical Smad signaling and non-canonical signaling, including the Ras-induced ERK pathway (Raf–MEK–ERK). However, the signaling mechanisms regulating TGF-β1-induced EMT are not fully understood. Here, we show that the Ras–ERK pathway negatively regulates TGF-β1-induced EMT in RLE-6TN cells and that DA-Raf1 (DA-Raf), a splicing isoform of A-Raf and a dominant-negative antagonist of the Ras–ERK pathway, plays an essential role in EMT. Stimulation of the cells with fibroblast growth factor 2 (FGF2), which activated the ERK pathway, prominently suppressed TGF-β1-induced EMT. An inhibitor of MEK, but not an inhibitor of phosphatidylinositol 3-kinase, rescued the TGF-β1-treated cells from the suppression of EMT by FGF2. Overexpression of a constitutively active mutant of a component of the Ras–ERK pathway, i.e., H-Ras, B-Raf, or MEK1, interfered with EMT. Knockdown of DA-Raf expression with siRNAs facilitated the activity of MEK and ERK, which were only weakly and transiently activated by TGF-β1. Although DA-Raf knockdown abrogated TGF-β1-induced EMT, the abrogation of EMT was reversed by the addition of the MEK inhibitor. Furthermore, DA-Raf knockdown impaired the TGF-β1-induced nuclear translocation of Smad2, which mediates the transcription required for EMT. These results imply that intrinsic DA-Raf exerts essential functions for EMT by antagonizing the TGF-β1-induced Ras–ERK pathway in RLE-6TN cells.</p></div

Suppression of the ERK pathway by DA-Raf is required for TGF-β1-induced EMT.

<p>(A) Induction of the binding of DA-Raf to Ras by TGF-β1 stimulation. The binding was analyzed by a coimmunoprecipitation assay. RLE cells were treated with 0.5 ng/ml TGF-β1 for 5 min. DA-Raf was immunoprecipitated with anti-DA-Raf pAb, and coprecipitated Ras was detected by immunoblotting with pan-Ras mAb. (B) Elevation of the phosphorylation levels of MEK and ERK by DA-Raf knockdown. RLE cells were transfected with <i>DAraf</i> siRNA1 as well as the control siRNA and treated with 0.5 ng/ml TGF-β1 for the indicated time. The levels of MEK, P-MEK, ERK, P-ERK, DA-Raf, A-Raf, and β-tubulin were analyzed by immunoblotting. The relative intensities of P-MEK1/2 and P-ERK1/2 bands are indicated under their blots. (C) Recovery of <i>DAraf</i> siRNA-blocked αSMA expression with U0126. RLE cells were transfected with <i>DAraf</i> siRNA1 or siRNA2 and then treated with 2 or 5 μM U0126 and 0.5 ng/ml TGF-β1 for 48 h. The level of αSMA, as well as β-tubulin as a standard, was analyzed by immunoblotting. (D) Recovery of <i>DAraf</i> siRNA-impaired αSMA expressing cells with U0126. RLE cells were transfected with <i>DAraf</i> siRNA1 or siRNA2 and then treated with 5 μM U0126 and 0.5 ng/ml TGF-β1 for 48 h. αSMA expression (red) and nuclei (blue) were detected. Scale bar, 50 μm. (E) The ratio of αSMA-expressing cells in the analysis of (D). The values are means ± SD of 3 independent experiments. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; #, <i>P</i> > 0.05 (not significant) by <i>t</i> test.</p

Knockdown of DA-Raf abrogates TGF-β1-induced EMT.

<p>(A) Suppression of TGF-β1-induced αSMA expression by knockdown of DA-Raf with <i>DAraf</i> siRNAs. RLE cells were transfected with <i>DAraf</i> siRNAs as well as a control siRNA. Twenty-four hours after the transfection, they were treated with 0.5 ng/ml TGF-β1 for 48 h. The levels of DA-Raf, A-Raf, αSMA, and E-cadherin, as well as β-tubulin as a standard, were analyzed by immunoblotting. (B) Suppression of TGF-β1-induced αSMA expression with <i>DAraf</i> siRNAs. RLE cells were transfected with <i>DAraf</i> siRNAs and treated with 0.5 ng/ml TGF-β1. αSMA expression (red) and nuclei (blue) were detected by fluorescence microscopy. Scale bar, 50 μm. (C) The ratio of αSMA-expressing cells in the analysis of (B). The values are means ± SD of 3 independent experiments. **, <i>P</i> < 0.01 by <i>t</i> test. (D) Elevation of the TGF-β1-suppressed <i>Cdh1</i> (E-cadherin) mRNA level and suppression of the TGF-β1-induced <i>Acta2</i> (αSMA) mRNA level with <i>DAraf</i> siRNAs. RLE cells were transfected with <i>DAraf</i> siRNAs and treated with TGF-β1 for 48 h. Relative levels of <i>Cdh1</i> and <i>Acta2</i> mRNAs normalized to the <i>Actb</i> (β-actin) mRNA level were determined by real-time PCR. The values are means ± SD of 3 independent experiments. **, <i>P</i> < 0.01 by <i>t</i> test. (E) Dose-dependent induction of αSMA expression by TGF-β1 and its suppression by <i>DAraf</i> siRNAs. RLE cells were transfected with <i>DAraf</i> siRNAs and treated with 0.1–5 ng/ml TGF-β1 for 48 h. The intensities of αSMA and β-tubulin bands on immunoblots were analyzed by densitometry. The graph shows the ratio of αSMA to β-tubulin band intensity against TGF-β1 concentration. The values are means ± SD of 3 independent experiments. a.u., arbitrary units.</p

FGF2 induces sustained activation of the Ras—ERK pathway and inhibits TGF-β1-induced EMT.

<p>(A) Transient phosphorylation of MEK and ERK and induction of αSMA expression by TGF-β1 stimulation. RLE cells were stimulated with 0.5 ng/ml TGF-β1. The levels of MEK, phospho (P)-MEK, ERK, P-ERK, and αSMA, as well as β -tubulin as a standard, were analyzed by immunoblotting. (B) Sustained phosphorylation of MEK and ERK and inhibition of αSMA expression by FGF2 stimulation. RLE cells were stimulated with 100 ng/ml FGF2 in combination with 0.5 ng/ml TGF-β1. (C) A dose-dependent reduction of the TGF- β 1-induced αSMA protein level by FGF2 stimulation. RLE cells were stimulated with the indicated concentrations of FGF2 together with 0.5 ng/ml TGF- β 1 for 48 h. The relative intensity of αSMA band is indicated under the blot. (D) Induction of αSMA expression by TGF- β 1 and suppression of the expression by FGF2. RLE cells were stimulated with 0.5 ng/ml TGF- β 1 or with 100 ng/ml FGF2 along with TGF- β 1 for 48 h. αSMA expression and localization was detected by immunofluorescent staining with the Cy3—anti-αSMA mAb (red) as well as nuclear staining with Hoechst 33258 (blue). Scale bar, 50 μm. (E) A dose-dependent reduction of the ratio of TGF- β 1-induced αSMA-expressing cells by FGF2 stimulation. αSMA-expressing cells were detected as in (D). The values are means ± SD of 3 independent experiments. **, <i>P</i> < 0.01 by <i>t</i> test.</p

Knockdown of DA-Raf hinders the nuclear translocation of Smad2 induced by TGF-β1.

<p>(A) Live cell images of the localization of Smad2. mCherry—Smad2-expressing RLE cells transfected with the control siRNA or <i>DAraf</i> siRNA1 were stimulated with 0.5 ng/ml TGF-β1 for the indicated time. The color indicator shows fluorescence intensity of mCherry—Smad2. Scale bar, 20 μm. (B) The degree of mCherry—Smad2 localization in the nucleus in the analysis of (A). Smad2 localized to the nucleus was calculated from the nuclear/cytoplasmic ratio of mCherry—Smad2 intensity. The box plot represents the data of 4 independent live cell images. *, <i>P</i> < 0.03; **, <i>P</i> < 0.005 by <i>t</i> test.</p

Inhibition of MEK but not PI3K recovers TGF-β1-induced and FGF2-suppressed EMT.

<p>(A) Recovery of FGF2-suppressed αSMA expression by MEK inhibition but not by PI3K inhibition. RLE cells were pretreated with 10 μM of the MEK inhibitor U0126 or the PI3K inhibitor LY294002 for 30 min. Then they were stimulated with 0.5 ng/ml TGF-β1 along with 100 ng/ml FGF2 for 48 h. αSMA expression (red) and nuclei (blue) were detected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127888#pone.0127888.g001" target="_blank">Fig 1</a> legend. Scale bar, 50 μm. (B) The ratio of αSMA-expressing cells in the analysis of (A). The values are means ± SD of 3 independent experiments. **, <i>P</i> < 0.01; #, <i>P</i> > 0.05 (not significant) by <i>t</i> test.</p