Cirrhosis results from accumulation of myofibroblasts derived from quiescent hepatic stellate cells (Q-HSCs); it regresses when myofibroblastic HSCs are depleted. Hedgehog signaling promotes ...transdifferentiation of HSCs by activating Yes-associated protein 1 (YAP1 or YAP) and inducing aerobic glycolysis. However, increased aerobic glycolysis alone cannot meet the high metabolic demands of myofibroblastic HSCs. Determining the metabolic processes of these cells could lead to strategies to prevent progressive liver fibrosis, so we investigated whether glutaminolysis (conversion of glutamine to alpha-ketoglutarate) sustains energy metabolism and permits anabolism when Q-HSCs become myofibroblastic, and whether this is controlled by hedgehog signaling to YAP.
Primary HSCs were isolated from C57BL/6 or Smoflox/flox mice; we also performed studies with rat and human myofibroblastic HSCs. We measured changes of glutaminolytic genes during culture-induced primary HSC transdifferentiation. Glutaminolysis was disrupted in cells by glutamine deprivation or pathway inhibitors (bis-2-5-phenylacetamido-1,2,4-thiadiazol-2-yl ethyl sulfide, CB-839, epigallocatechin gallate, and aminooxyacetic acid), and effects on mitochondrial respiration, cell growth and migration, and fibrogenesis were measured. Hedgehog signaling to YAP was disrupted in cells by adenovirus expression of Cre-recombinase or by small hairpin RNA knockdown of YAP. Hedgehog and YAP activity were inhibited by incubation of cells with cyclopamine or verteporfin, and effects on glutaminolysis were measured. Acute and chronic liver fibrosis were induced in mice by intraperitoneal injection of CCl4 or methionine choline-deficient diet. Some mice were then given injections of bis-2-5-phenylacetamido-1,2,4-thiadiazol-2-yl ethyl sulfide to inhibit glutaminolysis, and myofibroblast accumulation was measured. We also performed messenger RNA and immunohistochemical analyses of percutaneous liver biopsies from healthy human and 4 patients with no fibrosis, 6 patients with mild fibrosis, and 3 patients with severe fibrosis.
Expression of genes that regulate glutaminolysis increased during transdifferentiation of primary Q-HSCs into myofibroblastic HSCs, and inhibition of glutaminolysis disrupted transdifferentiation. Blocking glutaminolysis in myofibroblastic HSCs suppressed mitochondrial respiration, cell growth and migration, and fibrogenesis; replenishing glutaminolysis metabolites to these cells restored these activities. Knockout of the hedgehog signaling intermediate smoothened or knockdown of YAP inhibited expression of glutaminase, the rate-limiting enzyme in glutaminolysis. Hedgehog and YAP inhibitors blocked glutaminolysis and suppressed myofibroblastic activities in HSCs. In livers of patients and of mice with acute or chronic fibrosis, glutaminolysis was induced in myofibroblastic HSCs. In mice with liver fibrosis, inhibition of glutaminase blocked accumulation of myofibroblasts and fibrosis progression.
Glutaminolysis controls accumulation of myofibroblast HSCs in mice and might be a therapeutic target for cirrhosis.
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Recent statements by United Nations bodies point to free, prior and informed consent (FPIC) as a potential requirement in the development of engineered gene drive applications. As a concept developed ...in the context of protecting Indigenous rights to self-determination in land development scenarios, FPIC would need to be extended to apply to the context of ecological editing. Without an explicit framework of application, FPIC could be interpreted as a narrowly framed process of community consultation focused on the social implications of technology, and award little formal or advisory power in decision-making to Indigenous peoples and local communities. In this paper, we argue for an articulation of FPIC that attends to issues of transparency, iterative community-scale consent, and shared power through co-development among Indigenous peoples, local communities, researchers and technology developers. In realizing a comprehensive FPIC process, researchers and developers have an opportunity to incorporate enhanced participation and social guidance mechanisms into the design, development and implementation of engineered gene drive applications.
The
gene encodes a type I single-pass transmembrane protein that contains a large extracellular domain, a membrane spanning segment, and a short intracellular domain. Klotho protein exists in several ...forms including the full-length membrane form (mKl) and a soluble circulating form soluble klotho (sKl). mKl complexes with fibroblast growth factor receptors to form coreceptors for FGF23, which allows it to participate in FGF23-mediated signal transduction and regulation of phosphate and calcium homeostasis. sKl is present in the blood, urine, and cerebrospinal fluid where it performs a multitude of functions including regulation of ion channels/transporters and growth factor signaling. How sKl exerts these pleiotropic functions is poorly understood. One hurdle in understanding sKl's mechanism of action as a "hormone" has been the inability to identify a receptor that mediates its effects. In the body, the kidneys are a major source of sKl and sKl levels decline during renal disease. sKl deficiency in chronic kidney disease makes the heart susceptible to stress-induced injury. Here, we summarize the current knowledge of mKl's mechanism of action, the mechanistic basis of sKl's protective, FGF23-independent effects on the heart, and provide new insights into the mechanism of action of sKl focusing on recent findings that sKl binds sialogangliosides in membrane lipid rafts to regulate growth factor signaling.
BACKGROUND AND PURPOSE
Signalling networks that regulate the progression of cannabinoid CB1 receptor‐mediated extracellular signal‐regulated kinase (ERK) activation in neurons are poorly understood. ...We investigated the cellular mechanisms involved in CB1 receptor‐stimulated ERK phosphorylation in a neuronal cell model.
EXPERIMENTAL APPROACH
Murine N18TG2 neuronal cells were used to analyse the effect of specific protein kinase and phosphatase inhibitors on CB1 receptor‐stimulated ERK phosphorylation. The LI‐COR In Cell Western assay and immunoblotting were used to measure ERK phosphorylation.
KEY RESULTS
The time‐course of CB1 receptor‐stimulated ERK activation occurs in three phases that are regulated by distinct cellular mechanisms in N18TG2 cells. Phase I (0–5 min) maximal ERK phosphorylation is mediated by CB1 receptor‐stimulated ligand‐independent transactivation of multiple receptor tyrosine kinases (RTKs). Phase I requires Gi/oβγ subunit‐stimulated phosphatidylinositol 3‐kinase activation and Src kinase activation and is modulated by inhibition of cAMP‐activated protein kinase A (PKA) levels. Src kinase activation is regulated by the protein tyrosine phosphatases 1B and Shp1. The Phase II (5–10 min) rapid decline in ERK phosphorylation involves PKA inhibition and serine/threonine phosphatase PP1/PP2A activation. The Phase III (>10 min) plateau in ERK phosphorylation is mediated by CB1 receptor‐stimulated, ligand‐independent, transactivation of multiple RTKs.
CONCLUSIONS AND IMPLICATIONS
The complex expression of CB1 receptor‐stimulated ERK activation provides cellular selectivity, modulation of sensitivity to agonists, and coincidence detection with RTK signalling. RTK and PKA pathways may provide routes to novel CB1‐based therapeutic interventions in the treatment of addictive disorders or neurodegenerative diseases.
LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue‐8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue‐7
While the research community continues to develop novel proposals for intrinsic biocontainment of genetically engineered organisms, translation to real-world deployment faces several challenges.
BACKGROUND AND PURPOSE
Signalling networks that regulate the progression of cannabinoid CB
1
receptor‐mediated extracellular signal‐regulated kinase (ERK) activation in neurons are poorly understood. ...We investigated the cellular mechanisms involved in CB
1
receptor‐stimulated ERK phosphorylation in a neuronal cell model.
EXPERIMENTAL APPROACH
Murine N18TG2 neuronal cells were used to analyse the effect of specific protein kinase and phosphatase inhibitors on CB
1
receptor‐stimulated ERK phosphorylation. The LI‐COR In Cell Western assay and immunoblotting were used to measure ERK phosphorylation.
KEY RESULTS
The time‐course of CB
1
receptor‐stimulated ERK activation occurs in three phases that are regulated by distinct cellular mechanisms in N18TG2 cells. Phase I (0–5 min) maximal ERK phosphorylation is mediated by CB
1
receptor‐stimulated ligand‐independent transactivation of multiple receptor tyrosine kinases (RTKs). Phase I requires G
i/o
βγ subunit‐stimulated phosphatidylinositol 3‐kinase activation and Src kinase activation and is modulated by inhibition of cAMP‐activated protein kinase A (PKA) levels. Src kinase activation is regulated by the protein tyrosine phosphatases 1B and Shp1. The Phase II (5–10 min) rapid decline in ERK phosphorylation involves PKA inhibition and serine/threonine phosphatase PP1/PP2A activation. The Phase III (>10 min) plateau in ERK phosphorylation is mediated by CB
1
receptor‐stimulated, ligand‐independent, transactivation of multiple RTKs.
CONCLUSIONS AND IMPLICATIONS
The complex expression of CB
1
receptor‐stimulated ERK activation provides cellular selectivity, modulation of sensitivity to agonists, and coincidence detection with RTK signalling. RTK and PKA pathways may provide routes to novel CB
1
‐based therapeutic interventions in the treatment of addictive disorders or neurodegenerative diseases.
LINKED ARTICLES
This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit
http://dx.doi.org/10.1111/bph.2012.165.issue‐8
. To view Part I of Cannabinoids in Biology and Medicine visit
http://dx.doi.org/10.1111/bph.2011.163.issue‐7
Soluble klotho, the shed ectodomain of the antiaging membrane protein α-klotho, is a pleiotropic endocrine/paracrine factor with no known receptors and poorly understood mechanism of action. Soluble ...klotho down-regulates growth factor-driven PI3K signaling, contributing to extension of lifespan, cardioprotection, and tumor inhibition. Here we show that soluble klotho binds membrane lipid rafts. Klotho binding to rafts alters lipid organization, decreases membrane’s propensity to form large ordered domains for endocytosis, and down-regulates raft-dependent PI3K/Akt signaling. We identify α2-3-sialyllactose present in the glycan of monosialogangliosides as targets of soluble klotho. α2-3-Sialyllactose is a common motif of glycans. To explain why klotho preferentially targets lipid rafts we show that clustering of gangliosides in lipid rafts is important. In vivo, raft-dependent PI3K signaling is up-regulated in klotho-deficient mouse hearts vs. wild-type hearts. Our results identify ganglioside-enriched lipid rafts to be receptors that mediate soluble klotho regulation of PI3K signaling. Targeting sialic acids may be a general mechanism for pleiotropic actions of soluble klotho.
Abstract Background Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with higher incidence in males and is characterized by atypical verbal/nonverbal communication, ...restricted interests that can be accompanied by repetitive behavior, and disturbances in social behavior. This study investigated brain mechanisms that contribute to sociability deficits and sex differences in an ASD animal model. Methods Sociability was measured in C58/J and C57BL/6J mice using the 3-chamber social choice test. Bulk RNA-Seq and snRNA-Seq identified transcriptional changes in C58/J and C57BL/6J amygdala within which DMRseq was used to measure differentially methylated regions in amygdala. Results C58/J mice displayed divergent social strata in the 3-chamber test. Transcriptional and pathway signatures revealed immune-related biological processes differ between C58/J and C57BL/6J amygdala. Hypermethylated and hypomethylated genes were identified in C58/J versus C57BL/6J amygdala. snRNA-Seq data in C58/J amygdala identified differential transcriptional signatures within oligodendrocytes and microglia characterized by increased ASD risk gene expression and predicted impaired myelination that was dependent on sex and sociability. RNA velocity, gene regulatory network, and cell communication analysis showed diminished oligodendrocyte/microglia differentiation. Findings were verified using Bulk RNA-Seq and demonstrated oxytocin’s beneficial effects on myelin gene expression. Limitations Our findings are significant. However, limitations can be noted. The cellular mechanisms linking reduced oligodendrocyte differentiation and reduced myelination to an ASD phenotype in C58/J mice need further investigation. Additional snRNA-Seq and spatial studies would determine if effects in oligodendrocytes/microglia are unique to amygdala or if this occurs in other brain regions. Oxytocin’s effects need further examination to understand its’ potential as an ASD therapeutic. Conclusions Our work demonstrates the C58/J mouse model’s utility in evaluating the influence of sex and sociability on the transcriptome in concomitant brain regions involved in ASD. Our single-nucleus transcriptome analysis elucidates potential pathological roles of oligodendrocytes and microglia in ASD. This investigation provides details regarding regulatory features disrupted in these cell types, including transcriptional gene dysregulation, aberrant cell differentiation, altered gene regulatory networks, and changes to key pathways that promote microglia/oligodendrocyte differentiation. Our studies provide insight into interactions between genetic risk and epigenetic processes associated with divergent affiliative behavior and lack of positive sociability.
Tyrosine phosphorylation (Tyr-P) of focal adhesion kinase (FAK) regulates FAK activation. Phosphorylated FAK Tyr 397 binds Src family kinases (Src), which in turn directly phosphorylate FAK Tyr ...576/577 to produce maximal FAK enzymatic activity. CB1 cannabinoid receptors (CB1) are abundantly expressed in the nervous system and influence FAK activation by presently unknown mechanisms. The current investigation determined that CB1-stimulated maximal FAK catalytic activity is mediated by Gi/o proteins in N18TG2 neuronal cells, and that G12/13 regulation of Rac1 and RhoA occurs concomitantly. Immunoblotting analyses using antibodies against FAK phospho-Tyr 397 and phospho-Tyr 576/577 demonstrated that the time-course of CB1-stimulated FAK 576/577 Tyr-P occurred in three phases: Phase I (0–2min) maximal Tyr-P, Phase II (5–20min) rapid decline in Tyr-P, and Phase III (>20min) plateau in Tyr-P at submaximal levels. In contrast, FAK 397 Tyr-P was monophasic and significantly lower in magnitude. FAK 397 Tyr-P and Phase I FAK 576/577 Tyr-P involved protein tyrosine phosphatase (PTP1B and Shp1/Shp2)-mediated Src activation, Protein Kinase A (PKA) inhibition, and integrin activation. Phase I maximal FAK 576/577 Tyr-P also required cooperative signaling between receptor tyrosine kinases (RTKs) and integrins. The integrin antagonist RGDS peptide, Flk-1 vascular endothelial growth factor receptor (VEGFR) antagonist SU5416, and epidermal growth factor receptor (EGFR) antagonist AG 1478 blocked Phase I FAK 576/577 Tyr-P. CB1 agonists failed to stimulate FAK Tyr-P in the absence of integrin activation upon suspension in serum-free culture media. In contrast, cells grown on the integrin ligands fibronectin and laminin displayed increased FAK 576/577 Tyr-P that was augmented by CB1 agonists and blocked by the Src inhibitor PP2 and Flk-1 VEGFR antagonist SU5416. Taken together, these studies have identified a complex integrative pathway utilized by CB1 to stimulate maximal FAK 576/577 Tyr-P in neuronal cells.
•A novel pathway triggering CB1-stimulated FAK activity was elucidated in neurons.•CB1 stimulated FAK 576/577 tyrosine phosphorylation in a triphasic manner.•CB1-stimulated FAK 576/577 tyrosine phosphorylation was adhesion-dependent.•Maximal FAK activation required cooperative signaling between integrins and RTKs.•Maximal FAK activation involved tyrosine phosphatase-mediated Src activation.
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