Glycogen synthase kinase-3 is essential for β-arrestin-2 complex formation and lithium-sensitive behaviors in mice

Lithium is the first-line therapy for bipolar disorder. However, its therapeutic target remains controversial. Candidates include inositol monophosphatases, glycogen synthase kinase-3 (GSK-3), and a β-arrestin-2/AKT/protein phosphatase 2A (β-arrestin-2/AKT/PP2A) complex that is known to be required for lithium-sensitive behaviors. Defining the direct target(s) is critical for the development of new therapies and for elucidating the molecular pathogenesis of this major psychiatric disorder. Here, we show what we believe to be a new link between GSK-3 and the β-arrestin-2 complex in mice and propose an integrated mechanism that accounts for the effects of lithium on multiple behaviors. GSK-3β (Gsk3b) overexpression reversed behavioral defects observed in lithium-treated mice and similar behaviors observed in Gsk3b+/- mice. Furthermore, immunoprecipitation of striatial tissue from WT mice revealed that lithium disrupted the β-arrestin-2/Akt/PP2A complex by directly inhibiting GSK-3. GSK-3 inhibitors or loss of one copy of the Gsk3b gene reduced β-arrestin-2/Akt/PP2A complex formation in mice, while overexpression of Gsk3b restored complex formation in lithium-treated mice. Thus, GSK-3 regulates the stability of the β-arrestin-2/Akt/PP2A complex, and lithium disrupts the complex through direct inhibition of GSK-3. We believe these findings reveal a new role for GSK-3 within the β-arrestin complex and demonstrate that GSK-3 is a critical target of lithium in mammalian behaviors.     

β₁Integrin/FAK/cortactin signaling is essential for human head and neck cancer resistance to radiotherapy

Integrin signaling critically contributes to the progression, growth, and therapy resistance of malignant tumors. Here, we show that targeting of β₁ integrins with inhibitory antibodies enhances the sensitivity to ionizing radiation and delays the growth of human head and neck squamous cell carcinoma cell lines in 3D cell culture and in xenografted mice. Mechanistically, dephosphorylation of focal adhesion kinase (FAK) upon inhibition of β₁ integrin resulted in dissociation of a FAK/cortactin protein complex. This, in turn, downregulated JNK signaling and induced cell rounding, leading to radiosensitization. Thus, these findings suggest that robust and selective pharmacological targeting of β₁ integrins may provide therapeutic benefit to overcome tumor cell resistance to radiotherapy.     

EWS/ATF1 expression induces sarcomas from neural crest–derived cells in mice

Clear cell sarcoma (CCS) is an aggressive soft tissue malignant tumor characterized by a unique t(12;22) translocation that leads to the expression of a chimeric EWS/ATF1 fusion gene. However, little is known about the mechanisms underlying the involvement of EWS/ATF1 in CCS development. In addition, the cellular origins of CCS have not been determined. Here, we generated EWS/ATF1-inducible mice and examined the effects of EWS/ATF1 expression in adult somatic cells. We found that forced expression of EWS/ATF1 resulted in the development of EWS/ATF1-dependent sarcomas in mice. The histology of EWS/ATF1-induced sarcomas resembled that of CCS, and EWS/ATF1-induced tumor cells expressed CCS markers, including S100, SOX10, and MITF. Lineage-tracing experiments indicated that neural crest–derived cells were subject to EWS/ATF1-driven transformation. EWS/ATF1 directly induced Fos in an ERK-independent manner. Treatment of human and EWS/ATF1-induced CCS tumor cells with FOS-targeted siRNA attenuated proliferation. These findings demonstrated that FOS mediates the growth of EWS/ATF1-associated sarcomas and suggest that FOS is a potential therapeutic target in human CCS.     

Activation of mTORC1 is essential for β-adrenergic stimulation of adipose browning

A classic metabolic concept posits that insulin promotes energy storage and adipose expansion, while catecholamines stimulate release of adipose energy stores by hydrolysis of triglycerides through β-adrenergic receptor (βARs) and protein kinase A (PKA) signaling. Here, we have shown that a key hub in the insulin signaling pathway, activation of p70 ribosomal S6 kinase (S6K1) through mTORC1, is also triggered by PKA activation in both mouse and human adipocytes. Mice with mTORC1 impairment, either through adipocyte-specific deletion of Raptor or pharmacologic rapamycin treatment, were refractory to the well-known βAR-dependent increase of uncoupling protein UCP1 expression and expansion of beige/brite adipocytes (so-called browning) in white adipose tissue (WAT). Mechanistically, PKA directly phosphorylated mTOR and RAPTOR on unique serine residues, an effect that was independent of insulin/AKT signaling. Abrogation of the PKA site within RAPTOR disrupted βAR/mTORC1 activation of S6K1 without affecting mTORC1 activation by insulin. Conversely, a phosphomimetic RAPTOR augmented S6K1 activity. Together, these studies reveal a signaling pathway from βARs and PKA through mTORC1 that is required for adipose browning by catecholamines and provides potential therapeutic strategies to enhance energy expenditure and combat metabolic disease.     

A novel OXA-10–like β-lactamase is present in different Enterobacteriaceae

OXA 101, a novel OXA-10 like enzyme, was found forming part of a class 1 integron located in a conjugative plasmid in three different species of Enterobacteriaceae. This β-lactamase is related to OXA-35 and OXA-56 and displays a narrow substrate hydrolysis profile.     

Location,location, regulation: a novel role for β-spectrin in the heart

Voltage-gated Na⁺ channels (VGSCs) are responsible for the rising phase of the action potential in excitable cells, including neurons and skeletal and cardiac myocytes. Small alterations in gating properties can lead to severe changes in cellular excitability, as evidenced by the plethora of heritable conditions attributed to mutations in VGSCs highlighting the need to better understand VGSC regulation. In this issue of the JCI, Hund et al. identify the ability of a key structural protein, β_IV-spectrin, to bind and recruit Ca²⁺/calmodulin kinase II to the channel at a cellular location key to successful action potential initiation and propagation, where it can mediate function and excitability.     

A “Tric” to tighten cell-cell junctions in the cochlea for hearing

Tricellulin is a tricellular tight junction–associated membrane protein that controls movement of solutes at these specialized cell intersections. Mutations in the gene encoding tricellulin, TRIC, lead to nonsyndromic deafness. In this issue of the JCI, Nayak et al. created a gene-targeted knockin mouse in order to mimic the pathology of a human TRIC mutation. Deafness appears to be caused either by an increase in the K⁺ ion concentration around the basolateral surfaces of the outer hair cells or, alternatively, by an increase in small molecules such as ATP around the hair bundle, leading to cellular dysfunction and degeneration. Furthermore, the mice have features suggestive of syndromic hearing loss, which may have implications for care and treatment of patients harboring TRIC mutations. Millions worldwide suffer from a debilitating hearing loss (1). In many regions of the world in which health care conditions and public health are less developed or in populations with high rates of consanguinity, the number of people affected is extremely high. Understanding the mechanisms leading to hearing loss may help widen the current scope of therapeutic options, which are currently restricted to hearing aids and cochlear implants. The causes of hearing loss are manifold, including both genetic and environmental factors. Hereditary hearing loss is caused by mutations in a wide variety of genes that encode the proteins associated with the transduction of sound waves from the external ear to the middle and inner ear and finally to the brain. To date, hereditary hearing loss has been linked to over 60 genes (2). The cochlea, located in the inner ear, is responsible for the conversion of sound to a neural electric excitatory signal (3). Two distinct fluids, the endolymph and perilymph, are contained within the cochlea. While the perilymph has an ionic composition similar to that of the general extracellular fluid, the endolymph is characterized by a high K⁺ concentration and endocochlear potential (EP), which are essential for hearing (4). An epithelial cellular sheet covering the cochlea creates a barrier that allows these fluids to maintain their composition.     

Inactivation of Wnt/β-catenin signaling in human adipose-derived stem cells is necessary for chondrogenic differentiation and maintenance

The Wnt/β-catenin signaling pathway plays critical roles in self-renewal and differentiation of mesenchymal stem cells. However, very little is known about its role in the chondrogenesis of human adipose-derived stem cells (hADSCs). In this study, we analyzed protein expression of several key components of the Wnt/β-catenin signaling pathway using a 21-day in vitro model of hADSC chondrogenesis. Wnt1, β-catenin, and GSK3β levels increased sharply at day 12, peaked at day 18, and then declined. Expression of TCF1, a target gene of Wnt/β-catenin signaling, closely followed that of Wnt1. These results were consistent with changes in endonuclear β-catenin levels. Gene expression of the chondrocyte-specific markers, collagen type II (COL II), SOX9, and aggrecan, increased during hADSC chondrogenesis, peaked at day 12, and then declined. Adding a Wnt inhibitor (days 0–21) resulted in consistently elevated levels of COL II, SOX9, and aggrecan mRNA. In contrast, adding Wnt1 (days 0–21) to cultures led to sustained Wnt/β-catenin signaling over the 21 days and suppressed expression of chondrocyte-specific markers. Moreover, adding Wnt1 at late stages of differentiation (day 18) further diminished chondrocyte-specific marker expression. Together, these results showed that inactivation of Wnt/β-catenin signaling is needed for the progression of chondrogenesis and the maturation and phenotype maintenance of chondroid cells.     

α1G-dependent T-type Ca2+ current antagonizes cardiac hypertrophy through a NOS3-dependent mechanism in mice

In noncontractile cells, increases in intracellular Ca²⁺ concentration serve as a second messenger to signal proliferation, differentiation, metabolism, motility, and cell death. Many of these Ca²⁺-dependent regulatory processes operate in cardiomyocytes, although it remains unclear how Ca²⁺ serves as a second messenger given the high Ca²⁺ concentrations that control contraction. T-type Ca²⁺ channels are reexpressed in adult ventricular myocytes during pathologic hypertrophy, although their physiologic function remains unknown. Here we generated cardiac-specific transgenic mice with inducible expression of α1G, which generates Ca_v3.1 current, to investigate whether this type of Ca²⁺ influx mechanism regulates the cardiac hypertrophic response. Unexpectedly, α1G transgenic mice showed no cardiac pathology despite large increases in Ca²⁺ influx, and they were even partially resistant to pressure overload–, isoproterenol-, and exercise-induced cardiac hypertrophy. Conversely, α1G^–/– mice displayed enhanced hypertrophic responses following pressure overload or isoproterenol infusion. Enhanced hypertrophy and disease in α1G^–/– mice was rescued with the α1G transgene, demonstrating a myocyte-autonomous requirement of α1G for protection. Mechanistically, α1G interacted with NOS3, which augmented cGMP-dependent protein kinase type I activity in α1G transgenic hearts after pressure overload. Further, the anti-hypertrophic effect of α1G overexpression was abrogated by a NOS3 inhibitor and by crossing the mice onto the Nos3^–/– background. Thus, cardiac α1G reexpression and its associated pool of T-type Ca²⁺ antagonize cardiac hypertrophy through a NOS3-dependent signaling mechanism.     

PPARγ-induced cardiolipotoxicity in mice is ameliorated by PPARα deficiency despite increases in fatty acid oxidation

Excess lipid accumulation in the heart is associated with decreased cardiac function in humans and in animal models. The reasons are unclear, but this is generally believed to result from either toxic effects of intracellular lipids or excessive fatty acid oxidation (FAO). PPARγ expression is increased in the hearts of humans with metabolic syndrome, and use of PPARγ agonists is associated with heart failure. Here, mice with dilated cardiomyopathy due to cardiomyocyte PPARγ overexpression were crossed with PPARα-deficient mice. Surprisingly, this cross led to enhanced expression of several PPAR-regulated genes that mediate fatty acid (FA) uptake/oxidation and triacylglycerol (TAG) synthesis. Although FA oxidation and TAG droplet size were increased, heart function was preserved and survival improved. There was no marked decrease in cardiac levels of triglyceride or the potentially toxic lipids diacylglycerol (DAG) and ceramide. However, long-chain FA coenzyme A (LCCoA) levels were increased, and acylcarnitine content was decreased. Activation of PKCα and PKCδ, apoptosis, ROS levels, and evidence of endoplasmic reticulum stress were also reduced. Thus, partitioning of lipid to storage and oxidation can reverse cardiolipotoxicity despite increased DAG and ceramide levels, suggesting a role for other toxic intermediates such as acylcarnitines in the toxic effects of lipid accumulation in the heart.     

A Computational Positron Emission Tomography Simulation Model for Imaging β-Amyloid in Mice

Purpose We aimed to develop a computational simulation model for -amyloid (A) positron emission tomography (PET) imaging.Procedures Model parameters were set to reproduce levels of A within the PDAPP mouse. Pharmacokinetic curves of virtual tracers were computed and a PET detector simulator was configured for a commercially available preclinical PET-imaging system.Results We modeled the effects of A therapy and tracer affinity on the ability to differentiate A levels by PET. Varying affinity had a significant effect on the ability to quantitate A. Further, PET tracers for A monomers were more sensitive to the therapeutic reduction in A levels than total brain amyloid. Following therapy, the decrease in total brain A corresponded to the slow rate of change in total amyloid load as expected.Conclusions We have developed a first proof-of-concept A-PET simulation model that will be a useful tool in the interpretation of preclinical A imaging data and tracer development.