Association between C-reactive protein and adiposity in women.

Context: The link between C-reactive protein (CRP) and adiposity deserves to be further explored considering the controversial diabetogenic role of CRP. Objective: We explored the potential causal role of CRP on measures of adiposity. Design: We used a Mendelian randomization approach with the CRP and LEPR genes as instrumental variables in a cross-sectional Caucasian population-based study comprising 2526 men and 2836 women. Adiposity was measured using body mass index (BMI), fat and lean mass estimated by bioelectrical impedance, and waist circumference. Results: Log-transformed CRP explained by the rs7553007 SNP tagging the CRP gene was significantly associated with BMI (regression coefficient: 1.22 [0.18;2.25], P=0.02) and fat mass (2.67 [0.65;4.68], P=0.01), but not with lean mass in women, whereas no association was found in men. Log-transformed CRP explained by the rs1805096 LEPR SNP was also positively associated, although not significantly, with BMI or fat mass. The combined CRP-LEPR instrument explained 2.24% and 0.77% of CRP variance in women and in men, respectively. Log-transformed CRP explained by this combined instrument was significantly associated with BMI (0.98 [0.32;1.63], P=0.004), fat mass (2.07 [0.79;3.34], P=0.001) and waist (2.09 [0.39;3.78], P=0.01) in women, but not in men. Conclusion: Our data suggest that CRP is causally and positively related to BMI in women, and that this is mainly due to fat mass. Results on the combined CRP-LEPR instrument suggest that leptin may play a role in the causal association between CRP and adiposity in women. Results in men were not significant.

Job switching in ants: role of a kinase

Reproductive division of labour is a defining characteristic of eusociality in insect societies. The task of reproduction is performed by the fertile males and queens of the colony, while the non-fertile female worker caste performs all other tasks related to colony upkeep, foraging and nest defence. Division of labour, or polyethism, within the worker caste is organized such that specific tasks are performed by discrete groups of individuals. Ordinarily, workers of one group will not participate in the tasks of other groups making the groups of workers behaviourally distinct. In some eusocial species, this has led to the evolution of a remarkable diversity of subcaste morphologies within the worker caste, and a division of labour amongst the subcastes. This caste polyethism is best represented in many species of ants where a smaller-bodied minor subcaste typically performs foraging duties while larger individuals of the major subcaste are tasked with nest defence. Recent work suggests that polyethism in the worker caste is influenced by an evolutionarily conserved, yet diversely regulated, gene called foraging (for), which encodes a cGMP-dependent protein kinase (PKG). Additionally, flexibility in the activity of this enzyme allows for workers from one task group to assist the workers of other task groups in times of need during the colony's life.

Role of salt-inducible kinase 1 in the activation of MEF2-dependent transcription by BDNF.

Substantial evidence supports a role for myocyte enhancer factor 2 (MEF2)-mediated transcription in neuronal survival, differentiation and synaptic function. In developing neurons, it has been shown that MEF2-dependent transcription is regulated by neurotrophins. Despite these observations, little is known about the cellular mechanisms by which neurotrophins activate MEF2 transcriptional activity. In this study, we examined the role of salt-inducible kinase 1 (SIK1), a member of the AMP-activated protein kinase (AMPK) family, in the regulation of MEF2-mediated transcription by the neurotrophin brain-derived neurotrophic factor (BDNF). We show that BDNF increases the expression of SIK1 in primary cultures of rat cortical neurons through the extracellular signal-regulated kinase 1/2 (ERK1/2)-signaling pathway. In addition to inducing SIK1 expression, BDNF triggers the phosphorylation of SIK1 at Thr182 and its translocation from the cytoplasm to the nucleus of cortical neurons. The effects of BDNF on the expression, phosphorylation and, translocation of SIK1 are followed by the phosphorylation and nuclear export of histone deacetylase 5 (HDAC5). Blockade of SIK activity with a low concentration of staurosporine abolished BDNF-induced phosphorylation and nuclear export of HDAC5 in cortical neurons. Importantly, stimulation of HDAC5 phosphorylation and nuclear export by BDNF is accompanied by the activation of MEF2-mediated transcription, an effect that is suppressed by staurosporine. Consistent with these data, BDNF induces the expression of the MEF2 target genes Arc and Nur77, in a staurosporine-sensitive manner. In further support of the role of SIK1 in the regulation of MEF2-dependent transcription by BDNF, we found that expression of wild-type SIK1 or S577A SIK1, a mutated form of SIK1 which is retained in the nucleus of transfected cells, is sufficient to enhance MEF2 transcriptional activity in cortical neurons. Together, these data identify a previously unrecognized mechanism by which SIK1 mediates the activation of MEF2-dependent transcription by BDNF.

Cholesterol is the major component of native lipoproteins activating the p38 mitogen-activated protein kinases.

Elevated low-density lipoprotein (LDL) levels induce activation of the p38 mitogen-activated protein kinase (MAPK), a stress-activated protein kinase potentially participating in the development of atherosclerosis. The nature of the lipoprotein components inducing p38 MAPK activation has remained unclear however. We show here that both LDLs and high-density lipoproteins (HDLs) have the ability to stimulate the p38 MAPKs with potencies that correlate with their cholesterol content. Cholesterol solubilized in methyl-beta-cyclodextrin was sufficient to activate the p38 MAPK pathway. Liposomes made of phosphatidylcholine (PC) or sphingomyelin, the two main phospholipids found in lipoproteins, were unable to stimulate the p38 MAPKs. In contrast, PC liposomes loaded with cholesterol potently activated this pathway. Reducing the cholesterol content of LDL particles lowered their ability to activate the p38 MAPKs. Cell lines representative of the three main cell types found in blood vessels (endothelial cells, smooth muscle cells and fibroblasts) all activated their p38 MAPK pathway in response to LDLs or cholesterol-loaded PC liposomes. These results indicate that elevated cholesterol content in lipoproteins, as seen in hypercholesterolemia, favors the activation of the stress-activated p38 MAPK pathway in cells from the vessel wall, an event that might contribute to the development of atherosclerosis.

Islet-brain1/JNK-interacting protein-1 is required for early embryogenesis in mice.

Islet-brain1/JNK-interacting protein-1 (IB1/JIP-1) is a scaffold protein that organizes the JNK, MKK7, and MLK1 to allow signaling specificity. Targeted disruption of the gene MAPK8IP1 encoding IB1/JIP-1 in mice led to embryonic death prior to blastocyst implantation. In culture, no IB1/JIP-1(-/-) embryos were identified indicating that accelerated cell death occurred during the first cell cycles. IB1/JIP-1 expression was detected in unfertilized oocytes, in spermatozoa, and in different stages of embryo development. Thus, despite the maternal and paternal transmission of the IB1/JIP-1 protein, early transcription of the MAPK8IP1 gene is required for the survival of the fertilized oocytes.

Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4.

In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI.

Signal transduction by the cloned glucagon-like peptide-1 receptor: comparison with signaling by the endogenous receptors of beta cell lines.

Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that potentiates glucose-induced insulin secretion by pancreatic beta cells. The mechanisms of interaction between GLP-1 and glucose signaling pathways are not well understood. Here we studied the coupling of the cloned GLP-1 receptor, expressed in fibroblasts or in COS cells, to intracellular second messengers and compared this signaling with that of the endogenous receptor expressed in insulinoma cell lines. Binding of GLP-1 to the cloned receptor stimulated formation of cAMP with the same dose dependence and similar kinetics, compared with the endogenous receptor of insulinoma cells. Compared with forskolin-induced cAMP accumulation, that induced by GLP-1 proceeded with the same initial kinetics but rapidly reached a plateau, suggesting fast desensitization of the receptor. Coupling to the phospholipase C pathway was assessed by measuring inositol phosphate production and variations in the intracellular calcium concentration. No GLP-1-induced production of inositol phosphates could be measured in the different cell types studied. A rise in the intracellular calcium concentration was nevertheless observed in transfected COS cells but was much smaller than that observed in response to norepinephrine in cells also expressing the alpha 1B-adrenergic receptor. Importantly, no such increase in the intracellular calcium concentration could be observed in transfected fibroblasts or insulinoma cells, which, however, responded well to thrombin or carbachol, respectively. Together, our data show that interaction between GLP-1 and glucose signaling pathways in beta cells may be mediated uniquely by an increase in the intracellular cAMP concentration, with the consequent activation of protein kinase A and phosphorylation of elements of the glucose-sensing apparatus or of the insulin granule exocytic machinery.

Placental growth factor-1 and epithelial haemato-retinal barrier breakdown: potential implication in the pathogenesis of diabetic retinopathy.

AIMS/HYPOTHESIS: Disruption of the retinal pigment epithelial (RPE) barrier contributes to sub-retinal fluid and retinal oedema as observed in diabetic retinopathy. High placental growth factor (PLGF) vitreous levels have been found in diabetic patients. This work aimed to elucidate the influence of PLGF-1 on a human RPE cell line (ARPE-19) barrier in vitro and on normal rat eyes in vivo. METHODS: ARPE-19 permeability was measured using transepithelial resistance and inulin flux under stimulation of PLGF-1, vascular endothelial growth factor (VEGF)-E and VEGF 165. Using RT-PCR, we evaluated the effect of hypoxic conditions or insulin on transepithelial resistance and on PLGF-1 and VEGF receptors. The involvement of mitogen-activated protein kinase (MEK, also known as MAPK)/extracellular signal-regulated kinase (ERK, also known as EPHB2) signalling pathways under PLGF-1 stimulation was evaluated by western blot analysis and specific inhibitors. The effect of PLGF-1 on the external haemato-retinal barrier was evaluated after intravitreous injection of PLGF-1 in the rat eye; evaluation was by semi-thin analysis and zonula occludens-1 immunolocalisation on flat-mounted RPE. RESULTS: In vitro, PLGF-1 induced a reversible decrease of transepithelial resistance and enhanced tritiated inulin flux. These effects were specifically abolished by an antisense oligonucleotide directed at VEGF receptor 1. Exposure of ARPE-19 cells to hypoxic conditions or to insulin induced an upregulation of PLGF-1 expression along with increased transcellular permeability. The PLGF-1-induced RPE cell permeability involved the MEK signalling pathway. Injection of PLGF-1 in the rat eye vitreous induced an opening of the RPE tight junctions with subsequent sub-retinal fluid accumulation, retinal oedema and cytoplasm translocation of junction proteins. CONCLUSIONS/INTERPRETATION: Our results indicate that PLGF-1 may be a potential regulation target for the control of diabetic retinal and macular oedema.

Role of Mitogen-Activated Protein Kinases in Myocardial Ischemia-Reperfusion Injury during Heart Transplantation.

In solid organ transplantation, ischemia/reperfusion (IR) injury during organ procurement, storage and reperfusion is an unavoidable detrimental event for the graft, as it amplifies graft inflammation and rejection. Intracellular mitogen-activated protein kinase (MAPK) signaling pathways regulate inflammation and cell survival during IR injury. The four best-characterized MAPK subfamilies are the c-Jun NH2-terminal kinase (JNK), extracellular signal- regulated kinase-1/2 (ERK1/2), p38 MAPK, and big MAPK-1 (BMK1/ERK5). Here, we review the role of MAPK activation during myocardial IR injury as it occurs during heart transplantation. Most of our current knowledge regarding MAPK activation and cardioprotection comes from studies of preconditioning and postconditioning in nontransplanted hearts. JNK and p38 MAPK activation contributes to myocardial IR injury after prolonged hypothermic storage. p38 MAPK inhibition improves cardiac function after cold storage, rewarming and reperfusion. Small-molecule p38 MAPK inhibitors have been tested clinically in patients with chronic inflammatory diseases, but not in transplanted patients, so far. Organ transplantation offers the opportunity of starting a preconditioning treatment before organ procurement or during cold storage, thus modulating early events in IR injury. Future studies will need to evaluate combined strategies including p38 MAPK and/or JNK inhibition, ERK1/2 activation, pre- or postconditioning protocols, new storage solutions, and gentle reperfusion.

Cellular mechanisms underlying the regulation of dendritic development by hepatocyte growth factor.

Acquisition of a mature dendritic morphology is critical for neural information processing. In particular, hepatocyte growth factor (HGF) controls dendritic arborization during brain development. However, the cellular mechanisms underlying the effects of HGF on dendritic growth remain elusive. Here, we show that HGF increases dendritic length and branching of rat cortical neurons through activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Activation of MAPK by HGF leads to the rapid and transient phosphorylation of cAMP response element-binding protein (CREB), a key step necessary for the control of dendritic development by HGF. In addition to CREB phosphorylation, regulation of dendritic growth by HGF requires the interaction between CREB and CREB-regulated transcription coactivator 1 (CRTC1), as expression of a mutated form of CREB unable to bind CRTC1 completely abolished the effects of HGF on dendritic morphology. Treatment of cortical neurons with HGF in combination with brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family that regulates dendritic development via similar mechanisms, showed additive effects on MAPK activation, CREB phosphorylation and dendritic growth. Collectively, these results support the conclusion that regulation of cortical dendritic morphology by HGF is mediated by activation of the MAPK pathway, phosphorylation of CREB and interaction of CREB with CRTC1.

Specific targeting of pro-death NMDA receptor signals with differing reliance on the NR2B PDZ ligand.

NMDA receptors (NMDARs) mediate ischemic brain damage, for which interactions between the C termini of NR2 subunits and PDZ domain proteins within the NMDAR signaling complex (NSC) are emerging therapeutic targets. However, expression of NMDARs in a non-neuronal context, lacking many NSC components, can still induce cell death. Moreover, it is unclear whether targeting the NSC will impair NMDAR-dependent prosurvival and plasticity signaling. We show that the NMDAR can promote death signaling independently of the NR2 PDZ ligand, when expressed in non-neuronal cells lacking PSD-95 and neuronal nitric oxide synthase (nNOS), key PDZ proteins that mediate neuronal NMDAR excitotoxicity. However, in a non-neuronal context, the NMDAR promotes cell death solely via c-Jun N-terminal protein kinase (JNK), whereas NMDAR-dependent cortical neuronal death is promoted by both JNK and p38. NMDAR-dependent pro-death signaling via p38 relies on neuronal context, although death signaling by JNK, triggered by mitochondrial reactive oxygen species production, does not. NMDAR-dependent p38 activation in neurons is triggered by submembranous Ca(2+), and is disrupted by NOS inhibitors and also a peptide mimicking the NR2B PDZ ligand (TAT-NR2B9c). TAT-NR2B9c reduced excitotoxic neuronal death and p38-mediated ischemic damage, without impairing an NMDAR-dependent plasticity model or prosurvival signaling to CREB or Akt. TAT-NR2B9c did not inhibit JNK activation, and synergized with JNK inhibitors to ameliorate severe excitotoxic neuronal loss in vitro and ischemic cortical damage in vivo. Thus, NMDAR-activated signals comprise pro-death pathways with differing requirements for PDZ protein interactions. These signals are amenable to selective inhibition, while sparing synaptic plasticity and prosurvival signaling.

RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification.

JNK Inhibition Reduced Retinal Ganglion Cell Death after Ischemia/Reperfusion In Vivo and after Hypoxia In Vitro.

Mitogen-activated protein kinases (MAPKs) are key regulators that have been linked to cell survival and death. Among the main classes of MAPKs, c-jun N-terminal kinase (JNK) has been shown to mediate cell stress responses associated with apoptosis. In Vitro, hypoxia induced a significant increase in 661W cell death that paralleled increased activity of JNK and c-jun. 661W cells cultured in presence of the inhibitor of JNK (D-JNKi) were less sensitive to hypoxia-induced cell death. In vivo, elevation in intraocular pressure (IOP) in the rat promoted cell death that correlated with modulation of JNK activation. In vivo inhibition of JNK activation with D-JNKi resulted in a significant and sustained decrease in apoptosis in the ganglion cell layer, the inner nuclear layer and the photoreceptor layer. These results highlight the protective effect of D-JNKi in ischemia/reperfusion induced cell death of the retina.