Src42 binding activity regulates Drosophila RAF by a novel CNK-dependent derepression mechanism

Connector enhancer of KSR (CNK), an essential component of Drosophila receptor tyrosine kinase/mitogen-activated protein kinase pathways, regulates oppositely RAF function. This bimodal property depends on the N-terminal region of CNK, which integrates RAS activity to stimulate RAF and a bipartite element, called the RAF-inhibitory region (RIR), which binds and inhibits RAF catalytic activity. Here, we show that the repressive effect of the RIR is counteracted by the ability of Src42 to associate, in an RTK-dependent manner, with a conserved region located immediately C-terminal to the RIR. Strikingly, we found that several cnk loss-of-function alleles have mutations clustered in this area and provide evidence that these mutations impair Src42 binding. Surprisingly, the derepressing effect of Src42 does not appear to involve its catalytic function, but critically depends on the ability of its SH3 and SH2 domains to associate with CNK. Together, these findings suggest that the integration of RTK-induced RAS and Src42 signals by CNK as a two-component input is essential for RAF activation in Drosophila.     

Anion transport regulates intracellular pH in renal cortical tissue

The regulation of cell pH by anion transport was examined in suspensions of rabbit renal proximal tubules. Values for cell pH were derived from 14C-labeled 5,5-dimethyloxazolidine-2,4-dione distribution. In buffer with 10 mM/l HCO3-- and gassed with 95% O2/5% CO2, the anion transport inhibitors, 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene and furosemide, raised the cell-to-extracellular pH gradient from 0.23 +/- 0.02 to 0.31 +/- 0.02 and 0.31 +/- 0.03, respectively, but in combination their effects were not additive. Replacement of extracellular Cl-- by NO3-- raised the pH gradient from 0.24 +/- 0.04 to 0.37 +/- 0.05. Neither inhibitor raised the pH gradient in Cl-- -free media. Incubation of suspensions in HCO3-- and CO2-free media raised the pH gradient from 0.18 +/- 0.02 to 0.29 +/- 0.03. Removal of Cl-- in addition to HCO3-- and CO2 raised the pH gradient still further, to 0.36 +/- 0.02. The results demonstrate that two different anion transport inhibitors raise cell pH and the cell-to-extracellular pH gradient in proximal tubules and are consistent with the idea that the mechanism for this effect is inhibition of alkali anion exit from the tubule cell. This process appears to depend on extracellular Cl-- and probably occurs primarily by HCO3-- transport. The results support the concept that alkali anion transport, most probably HCO3-- exit from the peritubular cell border, is an important regulator of cell pH in renal proximal tubule.     

Shaggy regulates tissue growth through Hippo pathway in Drosophila

Science China Life Sciences - The evolutionarily conserved Hippo pathway coordinates cell proliferation, differentiation and apoptosis to regulate organ growth and tumorigenesis. Hippo signaling...     

GDF1 is a novel mediator of macrophage infiltration in brown adipose tissue of obese mice

We previously demonstrated a marked upregulation in the bone morphogenic protein (BMP)/growth differentiation factor (GDF) family member, GDF5, which is capable of promoting brown adipogenesis, in brown adipose tissue (BAT) of obese mice. In this study, we identified other GDF family members, besides GDF5 that are responsive to different obesogenic signals in BAT using inborn and acquired obesity animal models. In BAT from leptin-deficient ob/ob mice, GDF1 expression was preferentially downregulated, whereas the expression of several other genes in the BMP/GDF family, including GDF5, was upregulated. Moreover, in cultured brown adipocytes exposed to tunicamycin and hydrogen peroxide, at concentrations not affecting cellular viability, GDF1 expression was significantly downregulated. Recombinant GDF1 failed to significantly alter brown adipogenesis, despite the promoted phosphorylation of Smad1/5/8 in cultured brown adipocytes, but accelerated Smad1/5/8 phosphorylation with a concomitant increase in the number of migrating cells during exposure in a manner sensitive to activin-like kinase inhibitors in macrophagic RAW264.7 cells. Similarly, accelerated migration was observed in murine peritoneal macrophages exposed to GDF1. These results indicate that obesity could lead to predominant downregulation of GDF1 expression in BAT, which can modulate cellular migration through a mechanism relevant to activation of the downstream Smad signaling pathway in adjacent macrophages.     

Geographical variation in resistance of the parasitoid Asobara tabids against encapsulation by Drosophila melanoqaster larvae: the mechanism explored

Abstract. The braconid parasitoid Asobara tabida Nees attacks larvae of several Drosophila species in fermenting substrates. Northwestern and central European populations of the parasitoid attack mainly D.subobscura Collin. Southern European parasitoids attack mainly D.melanogaster Meigen. Larvae of this last species can defend themselves against parasitoids by encapsulating the parasitoid egg. Parasitoids from southern European populations are better able to resist encapsulation of their eggs than their northwestern and central European conspecifics. The eggs of southern European parasitoids appear to have a 'sticky' egg chorion. As a result of this 'stickiness' the eggs become embedded in host tissue where they are not completely covered by the host's blood cells. This leads to, at most, partial encapsulation of the egg. Parasitoid larvae can escape from partially closed capsules.     

PDX regulates inflammatory cell infiltration via resident macrophage in LPS‐induced lung injury

Inflammatory cell infiltration contributes to the pathogenesis of acute respiratory distress syndrome (ARDS). Protectin DX (PDX), an endogenous lipid mediator, shows anti‐inflammatory and proresolution bioactions. In vivo, the mice were intraperitoneally injected with PDX (0.1 µg/mouse) after intratracheal (1 mg/kg) or intraperitoneal (10 mg/kg) LPS administration. Flow cytometry was used to measure inflammatory cell numbers. Clodronate liposomes were used to deplete resident macrophages. RT‐PCR, and ELISA was used to measure MIP‐2, MCP‐1, TNF‐α and MMP9 levels. In vitro, sorted neutrophils, resident and recruited macrophages (1 × 10⁶) were cultured with 1 μg/mL LPS and/or 100 nmol/L PDX to assess the chemokine receptor expression. PDX attenuated LPS‐induced lung injury via inhibiting recruited macrophage and neutrophil recruitment through repressing resident macrophage MCP‐1, MIP‐2 expression and release, respectively. Finally, PDX inhibition of neutrophil infiltration and transmembrane was associated with TNF‐α/MIP‐2/MMP9 signalling pathway. These data suggest that PDX attenuates LPS‐stimulated lung injury via reduction of the inflammatory cell recruitment mediated via resident macrophages.     

Myeloid heme oxygenase-1 haploinsufficiency reduces high fat diet-induced insulin resistance by affecting adipose macrophage infiltration in mice

Increased adipose tissue macrophages contribute to obesity-induced metabolic syndrome. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with potent anti-inflammatory and proangiogenic activities in macrophages. However, the role of macrophage HO-1 on obesity-induced adipose inflammation and metabolic syndrome remains unclear. Here we show that high-fat diet (HFD) feeding in C57BL/6J mice induced HO-1 expression in the visceral adipose tissue, particularly the stromal vascular fraction. When the irradiated C57BL/6J mice reconstituted with wild-type or HO-1(+/-) bone marrow were fed with HFD for over 24 weeks, the HO-1(+/-) chimeras were protected from HFD-induced insulin resistance and this was associated with reduced adipose macrophage infiltration and angiogenesis, suggesting that HO-1 affects myeloid cell migration toward adipose tissue during obesity. In vivo and in vitro migration assays revealed that HO-1(+/-) macrophages exhibited an impaired migration response. Chemoattractant-induced phosphorylation of p38 and focal adhesion kinase (FAK) declined faster in HO-1(+/-) macrophages. Further experiments demonstrated that carbon monoxide and bilirubin, the byproducts derived from heme degradation by HO-1, enhanced macrophage migration by increasing phosphorylation of p38 and FAK, respectively. These data disclose a novel role of hematopoietic cell HO-1 in promoting adipose macrophage infiltration and the development of insulin resistance during obesity.     

Heteromerization of innexin gap junction proteins regulates epithelial tissue organization in Drosophila

Gap junctions consist of clusters of intercellular channels, which enable direct cell-to-cell communication and adhesion in animals. Whereas deuterostomes, including all vertebrates, use members of the connexin and pannexin multiprotein families to assemble gap junction channels, protostomes such as Drosophila and Caenorhabditis elegans use members of the innexin protein family. The molecular composition of innexin-containing gap junctions and the functional significance of innexin oligomerization for development are largely unknown. Here, we report that heteromerization of Drosophila innexins 2 and 3 is crucial for epithelial organization and polarity of the embryonic epidermis. Both innexins colocalize in epithelial cell membranes. Innexin3 is mislocalized to the cytoplasm in innexin2 mutants and is recruited into ectopic expression domains defined by innexin2 misexpression. Conversely, RNA interference (RNAi) knockdown of innexin3 causes mislocalization of innexin2 and of DE-cadherin, causing cell polarity defects in the epidermis. Biochemical interaction studies, surface plasmon resonance analysis, transgenesis, and biochemical fractionation experiments demonstrate that both innexins interact via their C-terminal cytoplasmic domains during the assembly of heteromeric channels. Our data provide the first molecular and functional demonstration that innexin heteromerization occurs in vivo and reveal insight into a molecular mechanism by which innexins may oligomerize into heteromeric gap junction channels.     

Naringenin suppresses macrophage infiltration into adipose tissue in an early phase of high-fat diet-induced obesity

Obese adipose tissue is characterized by increased macrophage infiltration, which results in chronic inflammation in adipose tissue and leads to obesity-related diseases such as type 2 diabetes mellitus and atherosclerosis. The regulation of macrophage infiltration into adipose tissue is an important strategy for preventing and treating obesity-related diseases. In this study, we report that naringenin, a citrus flavonoid, suppressed macrophage infiltration into adipose tissue induced by short-term (14 days) feeding of a high-fat diet in mice; although naringenin did not show any differences in high-fat diet-induced changes of serum biochemical parameters in this short administration period. Naringenin suppressed monocyte chemoattractant protein-1 (MCP-1) in adipose tissue, and this effect was mediated in part through inhibition of c-Jun NH₂-terminal kinase pathway. Naringenin also inhibited MCP-1 expression in adipocytes, macrophages, and a co-culture of adipocytes and macrophages. Our results suggest a mechanism by which daily consumption of naringenin may exhibit preventive effects on obesity-related diseases.     

aPKC regulates apical constriction to prevent tissue rupture in the Drosophila follicular epithelium

1. Download : Download high-res image (219KB)
2. Download : Download full-size image

     

Biphasic response as a mechanism against mutant takeover in tissue homeostasis circuits

Tissues use feedback circuits in which cells send signals to each other to control their growth and survival. We show that such feedback circuits are inherently unstable to mutants that misread the signal level: Mutants have a growth advantage to take over the tissue, and cannot be eliminated by known cell‐intrinsic mechanisms. To resolve this, we propose that tissues have biphasic responses in which the signal is toxic at both high and low levels, such as glucotoxicity of beta cells, excitotoxicity in neurons, and toxicity of growth factors to T cells. This gives most of these mutants a frequency‐dependent selective disadvantage, which leads to their elimination. However, the biphasic mechanisms create a new unstable fixed point in the feedback circuit beyond which runaway processes can occur, leading to risk of diseases such as diabetes and neurodegenerative disease. Hence, glucotoxicity, which is a dangerous cause of diabetes, may have a protective anti‐mutant effect. Biphasic responses in tissues may provide an evolutionary stable strategy that avoids invasion by commonly occurring mutants, but at the same time cause vulnerability to disease.     

Nebulin regulates actin filament lengths by a stabilization mechanism

Efficient muscle contraction requires regulation of actin filament lengths. In one highly cited model, the giant protein nebulin has been proposed to function as a molecular ruler specifying filament lengths. We directly challenged this hypothesis by constructing a unique, small version of nebulin (mini-nebulin). When endogenous nebulin was replaced with mini-nebulin in skeletal myocytes, thin filaments extended beyond the end of mini-nebulin, an observation which is inconsistent with a strict ruler function. However, under conditions that promote actin filament depolymerization, filaments associated with mini-nebulin were remarkably maintained at lengths either matching or longer than mini-nebulin. This indicates that mini-nebulin is able to stabilize portions of the filament it has no contact with. Knockdown of nebulin also resulted in more dynamic populations of thin filament components, whereas expression of mini-nebulin decreased the dynamics at both filament ends (i.e., recovered loss of endogenous nebulin). Thus, nebulin regulates thin filament architecture by a mechanism that includes stabilizing the filaments and preventing actin depolymerization.     

A dp53-dependent mechanism involved in coordinating tissue growth in Drosophila

Coordination of growth between and within organs contributes to the generation of well-proportioned organs and functionally integrated adults. The mechanisms that help to coordinate the growth between different organs start to be unravelled. However, whether an organ is able to respond in a coordinated manner to local variations in growth caused by developmental or environmental stress and the nature of the underlying molecular mechanisms that contribute to generating well-proportioned adult organs under these circumstances remain largely unknown. By reducing the growth rates of defined territories in the developing wing primordium of Drosophila, we present evidence that the tissue responds as a whole and the adjacent cell populations decrease their growth and proliferation rates. This non-autonomous response occurs independently of where growth is affected, and it is functional all throughout development and contributes to generate well-proportioned adult structures. Strikingly, we underscore a central role of Drosophila p53 (dp53) and the apoptotic machinery in these processes. While activation of dp53 in the growth-depleted territory mediates the non-autonomous regulation of growth and proliferation rates, effector caspases have a unique role, downstream of dp53, in reducing proliferation rates in adjacent cell populations. These new findings indicate the existence of a stress response mechanism involved in the coordination of tissue growth between adjacent cell populations and that tissue size and cell cycle proliferation can be uncoupled and are independently and non-autonomously regulated by dp53.     

Allelopathy of a native grassland community as a potential mechanism of resistance against invasion by introduced plants

Successful plant invasions depend, at least partly, on interactions between introduced plants and native plant communities. While allelopathic effects of introduced invaders on native resident species have received much attention, the reverse, i.e. allelopathic effects of native residents on introduced plants, have been largely neglected. Therefore, we tested whether allelopathy of native plant communities decreases their invasibility to introduced plant species. In addition, we tested among the introduced species whether the invasive ones are more tolerant to allelopathy of native plant communities than the non-invasive ones. To test these hypotheses, we grew nine pairs of related (congeneric or confamilial) invasive and non-invasive introduced plant species (i.e. 18 species) in the presence or absence of a native grassland community, which consisted of three common forbs and three common grasses, with or without activated carbon in the soil. Activated carbon reduced the survival percentage and growth of introduced plants in the absence of the native plant community. However, its net effect on the introduced plants was neutral or even slightly positive in the presence of the native community. This might suggest that the native plant community imposed allelopathic effects on the introduced plants, and that these effects were neutralized or reduced by activated carbon. The invasive and non-invasive introduced plants, however, did not differ in their tolerance to such allelopathic effects of the native plant community. Thus, although allelopathy of native plant communities might increase their resistance against introduced plants, there was no evidence that tolerance to allelopathy of native plant communities contributes to the degree of invasiveness of introduced plants.     

Dis3L2 regulates cell proliferation and tissue growth through a conserved mechanism

Dis3L2 is a highly conserved 3’-5’ exoribonuclease which is mutated in the human overgrowth disorders Perlman syndrome and Wilms’ tumour of the kidney. Using Drosophila melanogaster as a model system, we have generated a new dis3L2 null mutant together with wild-type and nuclease-dead genetic lines in Drosophila to demonstrate that the catalytic activity of Dis3L2 is required to control cell proliferation. To understand the cellular pathways regulated by Dis3L2 to control proliferation, we used RNA-seq on dis3L2 mutant wing discs to show that the imaginal disc growth factor Idgf2 is responsible for driving the wing overgrowth. IDGFs are conserved proteins homologous to human chitinase-like proteins such as CHI3L1/YKL-40 which are implicated in tissue regeneration as well as cancers including colon cancer and non-small cell lung cancer. We also demonstrate that loss of DIS3L2 in human kidney HEK-293T cells results in cell proliferation, illustrating the conservation of this important cell proliferation pathway. Using these human cells, we show that loss of DIS3L2 results in an increase in the PI3-Kinase/AKT signalling pathway, which we subsequently show to contribute towards the proliferation phenotype in Drosophila. Our work therefore provides the first mechanistic explanation for DIS3L2-induced overgrowth in humans and flies and identifies an ancient proliferation pathway controlled by Dis3L2 to regulate cell proliferation and tissue growth.     

ERp29 regulates response to doxorubicin by a PERK-mediated mechanism

ERp29 is an endoplasmic reticulum (ER) luminal protein with a putative secretion factor/escort chaperone function. Accumulated evidence has implicated ERp29 in the thyroglobulin secretion, polyoma virus transport and recently in carcinogenesis. ERp29 levels were elevated in the tumors of various origins and under the conditions of genotoxic stress, such as ionizing radiation. Here we report the induction of ERp29 during the treatment of cells with doxorubicin, a commonly used antineoplastic agent. Experiments in the p53 −/− cells and p53 knockout mouse revealed that doxorubicin effect on ERp29 is p53 dependent. The increase of ERp29 level appears to activate a negative feedback loop where the elevated amounts of ERp29 augment cell viability as shown by a clonogenic cell survival assay. To elucidate the mechanisms behind the doxorubicin effects we have studied the impact of ERp29 on the interaction with the ER stress-activated eukaryotic translation initiation factor 2-alpha kinase 3 (PERK) that was shown to facilitate tumor cells' resistance to drug toxicity. Co-immunoprecipitation demonstrated physical interaction of ERp29 with PERK and moreover, overexpression of ERp29 enhanced endogenous levels of PERK. Our results identify ERp29 as a novel regulator of PERK and provide evidence for the role of ER resident factors in the regulation of chemotherapeutic efficacy. These findings show that PERK may represent a nodal point between ER stress and chemotherapeutic response.