Receptor-mediated Intracellular Signaling Research Articles

Macrophage Function Modulated by tPA Signaling in Mouse Experimental Kidney Disease Models.

Macrophage infiltration and accumulation is a hallmark of chronic kidney disease. Tissue plasminogen activator (tPA) is a serine protease regulating the homeostasis of blood coagulation, fibrinolysis, and matrix degradation, and has been shown to act as a cytokine to trigger various receptor-mediated intracellular signal pathways, modulating macrophage function in response to kidney injury. In this review, we discuss the current understanding of tPA-modulated macrophage function and underlying signaling mechanisms during kidney fibrosis and inflammation.

Janus Kinase 3 phosphorylation and the JAK/STAT pathway are positively modulated by follicle-stimulating hormone (FSH) in bovine granulosa cells

Janus kinase 3 (JAK3) is a member of the JAK family of tyrosine kinase proteins involved in cytokine receptor-mediated intracellular signal transduction through the JAK/STAT signaling pathway. JAK3 was previously shown as differentially expressed in granulosa cells (GC) of bovine pre-ovulatory follicles suggesting that JAK3 could modulate GC function and activation/inhibition of downstream targets. We used JANEX-1, a JAK3 inhibitor, and FSH treatments and analyzed proliferation markers, steroidogenic enzymes and phosphorylation of target proteins including STAT3, CDKN1B/p27Kip1 and MAPK8IP3/JIP3. Cultured GC were treated with or without FSH in the presence or not of JANEX-1. Expression of steroidogenic enzyme CYP11A1, but not CYP19A1, was upregulated in GC treated with FSH and both were significantly decreased when JAK3 was inhibited. Proliferation markers CCND2 and PCNA were reduced in JANEX-1-treated GC and upregulated by FSH. Western blots analyses showed that JANEX-1 treatment reduced pSTAT3 amounts while JAK3 overexpression increased pSTAT3. Similarly, FSH treatment increased pSTAT3 even in JANEX-1-treated GC. UHPLC-MS/MS analyses revealed phosphorylation of specific amino acid residues within JAK3 as well as CDKN1B and MAPK8IP3 suggesting possible activation or inhibition post-FSH or JANEX-1 treatments. We show that FSH activates JAK3 in GC, which could phosphorylate target proteins and likely modulate other signaling pathways involving CDKN1B and MAPK8IP3, therefore controlling GC proliferation and steroidogenic activity.

Inhibitory effects of N-methyl-D-aspartate (NMDA) and α1-adrenergic receptor antagonist ifenprodil on human Kv1.5 channel.

Ifenprodil has been known to reduce cardiac contractility and cerebral vasodilation by antagonizing α1-adrenergic and N-methyl D-aspartate receptor-mediated intracellular signals. This study aimed to investigate the direct effect of ifenprodil on the human voltage-gated Kv1.5 channel (hKv1.5) by using a Xenopus oocyte expression system and a two-microelectrode voltage clamp technique. The amplitudes of hKv1.5 currents, including peak and steady state, were suppressed in a concentration-dependent manner (IC50; 43.1 and 35.5μM, respectively) after 6min of ifenprodil treatment. However, these effects were ~ 80% reversed by washout, suggesting that ifenprodil directly inhibited the hKv1.5 independent of membrane receptors or intracellular signals. The inhibition rate of steady state showed voltage dependence, wherein the rates increased according to test voltage depolarization. Ifenprodil reduced the time constants of hKv1.5 inactivation but has higher effects on activation. hKv1.5 inhibition by ifenprodil showed use dependency because the drug more rapidly reduced the current at the higher activation frequencies, and subsequent reduction in frequency after high activation frequency caused a partial channel block relief. Therefore, ifenprodil directly blocked the hKv1.5 in an open state and accelerated the time course of the channel inactivation, which provided a biophysical mechanism for the hKv1.5 blocking effects of ifenprodil.

Lyn attenuates sepsis-associated acute kidney injury by inhibition of phospho-STAT3 and apoptosis

Sepsis-associated acute kidney injury (SA-AKI) is a life-threatening condition associated with high mortality and morbidity. However, the underlying pathogenesis of SA-AKI is still unclear. Lyn belongs to Src family kinases (SFKs), which exert numerous biological functions including modulation in receptor-mediated intracellular signaling and intercellular communication. Previous studies demonstrated that Lyn gene deletion obviously aggravates LPS-induced lung inflammation, but the role and possible mechanism of Lyn in SA-AKI have not been reported yet. Here, we found that Lyn protected against renal tubular injury in cecal ligation and puncture (CLP) induced AKI mouse model by inhibition of signal transducer and activator of transcription 3 (STAT3) phosphorylation and cell apoptosis. Moreover, Lyn agonist MLR-1023 pretreatment improved renal function, inhibited STAT3 phosphorylation and decreased cell apoptosis. Thus, Lyn appears to play a crucial role in orchestrating STAT3-mediated inflammation and cell apoptosis in SA-AKI. Hence, Lyn kinase may be a promising therapeutic target for SA-AKI.

Striatal Subregion-selective Dysregulated Dopamine Receptor-mediated Intracellular Signaling in a Model of DOPA-responsive Dystonia

Although the mechanisms underlying dystonia are largely unknown, dystonia is often associated with abnormal dopamine neurotransmission. DOPA-responsive dystonia (DRD) is a prototype disorder for understanding dopamine dysfunction in dystonia because it is caused by mutations in genes necessary for the synthesis of dopamine and alleviated by the indirect-acting dopamine agonist l-DOPA. Although adaptations in striatal dopamine receptor-mediated intracellular signaling have been studied extensively in models of Parkinson’s disease, another movement disorders associated with dopamine deficiency, little is known about dopaminergic adaptations in dystonia. To identify the dopamine receptor-mediated intracellular signaling associated with dystonia, we used immunohistochemistry to quantify striatal protein kinase A activity and extracellular signal-related kinase (ERK) phosphorylation after dopaminergic challenges in a knockin mouse model of DRD. l-DOPA treatment induced the phosphorylation of both protein kinase A substrates and ERK largely in D1 dopamine receptor-expressing striatal neurons. As expected, this response was blocked by pretreatment with the D1 dopamine receptor antagonist SCH23390. The D2 dopamine receptor antagonist raclopride also significantly reduced the phosphorylation of ERK; this contrasts with models of parkinsonism in which l-DOPA-induced ERK phosphorylation is not mediated by D2 dopamine receptors. Further, the dysregulated signaling was dependent on striatal subdomains whereby ERK phosphorylation was largely confined to dorsomedial (associative) striatum while the dorsolateral (sensorimotor) striatum was unresponsive. This complex interaction between striatal functional domains and dysregulated dopamine-receptor mediated responses has not been observed in other models of dopamine deficiency, such as parkinsonism, suggesting that regional variation in dopamine-mediated neurotransmission may be a hallmark of dystonia.

Janus Kinase 3 phosphorylation and the JAK/STAT pathway are positively modulated by follicle‐stimulating hormone (FSH) in bovine granulosa cells

Janus kinase 3 (JAK3) is a member of the JAK family of tyrosine kinase proteins involved in cytokine receptor‐mediated intracellular signal transduction through the JAK/STAT signaling pathway. JAK3 was shown as differentially expressed in granulosa cells (GC) of bovine preovulatory follicles and downregulated by the luteinizing hormone. These observations suggested JAK3 regulation could modulate GC proliferation, steroidogenic activity and activation/inhibition of downstream targets. To investigate the mechanisms of JAK3 actions in GC, we used JANEX‐1, a pharmacological JAK3 inhibitor, and FSH treatments and analyzed proliferation markers, steroidogenic enzymes and phosphorylation of target proteins including STAT3 and previously identified JAK3 partners CDKN1B/p27Kip1 and MAPK8IP3/JIP3. Cultured GCs were treated with or without FSH in the presence or not of JANEX‐1. Total RNA and proteins were extracted and analyzed by RT‐qPCR, western blotting and UHPLC/MS‐MS. Expression of steroidogenic enzyme CYP11A1, but not CYP19A1, was significantly upregulated in GC treated with FSH and both were significantly decreased when JAK3 was inhibited as compared to control. Proliferation markers CCND2 and PCNA were significantly reduced in JANEX‐1‐treated GC and upregulated by FSH. Western blots analyses showed that JANEX‐1 treatment significantly reduced pSTAT3 amounts while JAK3 overexpression increased pSTAT3. Similarly, FSH treatment increased pSTAT3 even in JANEX‐1‐treated GC. UHPLC/MS‐MS analyses showed phosphorylation and additional modifications of specific amino acid residues within JAK3 as well as its binding partners CDKN1B and MAPK8IP3 revealing possible activation or inhibition of JAK3 following FSH or JANEX‐1 treatments, respectively. Abundance of JAK3 total protein was increased post‐FSH treatment and significantly decreased, along with MAPK8IP3, in JANEX‐1‐treated GC while CDKN1B total abundance was altered post‐FSH and increased post‐JANEX‐1. We show that JAK3 influences GC activity through phosphorylation of target proteins in response to stimulations such as FSH, which leads to the activation of JAK/STAT and likely modulating other signaling pathways involving CDKN1B and MAPK8IP3.

Elucidation of muscarinic receptor-mediated intracellular signaling aimed to restore, maintain, and promote salivary secretory activity

Elucidation of muscarinic receptor-mediated intracellular signaling aimed to restore, maintain, and promote salivary secretory activity

Immunomodulatory Effects of Vitamin D Supplementation in a Deficient Population

In addition to its canonical functions, vitamin D has been proposed to be an important mediator of the immune system. Despite ample sunshine, vitamin D deficiency is prevalent (>80%) in the Middle East, resulting in a high rate of supplementation. However, the underlying molecular mechanisms of the specific regimen prescribed and the potential factors affecting an individual’s response to vitamin D supplementation are not well characterized. Our objective is to describe the changes in the blood transcriptome and explore the potential mechanisms associated with vitamin D3 supplementation in one hundred vitamin D-deficient women who were given a weekly oral dose (50,000 IU) of vitamin D3 for three months. A high-throughput targeted PCR, composed of 264 genes representing the important blood transcriptomic fingerprints of health and disease states, was performed on pre and post-supplementation blood samples to profile the molecular response to vitamin D3. We identified 54 differentially expressed genes that were strongly modulated by vitamin D3 supplementation. Network analyses showed significant changes in the immune-related pathways such as TLR4/CD14 and IFN receptors, and catabolic processes related to NF-kB, which were subsequently confirmed by gene ontology enrichment analyses. We proposed a model for vitamin D3 response based on the expression changes of molecules involved in the receptor-mediated intra-cellular signaling pathways and the ensuing predicted effects on cytokine production. Overall, vitamin D3 has a strong effect on the immune system, G-coupled protein receptor signaling, and the ubiquitin system. We highlighted the major molecular changes and biological processes induced by vitamin D3, which will help to further investigate the effectiveness of vitamin D3 supplementation among individuals in the Middle East as well as other regions.

Innate Immunity in Diabetic Wound Healing: Focus on the Mastermind Hidden in Chronic Inflammatory.

A growing body of evidence suggests that the interaction between immune and metabolic responses is essential for maintaining tissue and organ homeostasis. These interacting disorders contribute to the development of chronic diseases associated with immune-aging such as diabetes, obesity, atherosclerosis, and nonalcoholic fatty liver disease. In Diabetic wound (DW), innate immune cells respond to the Pathogen-associated molecular patterns (PAMAs) and/or Damage-associated molecular patterns (DAMPs), changes from resting to an active phenotype, and play an important role in the triggering and maintenance of inflammation. Furthermore, the abnormal activation of innate immune pathways secondary to immune-aging also plays a key role in DW healing. Here, we review studies of innate immune cellular molecular events that identify metabolic disorders in the local microenvironment of DW and provide a historical perspective. At the same time, we describe some of the recent progress, such as TLR receptor-mediated intracellular signaling pathways that lead to the activation of NF-κB and the production of various pro-inflammatory mediators, NLRP3 inflammatory via pyroptosis, induction of IL-1β and IL-18, cGAS-STING responds to mitochondrial injury and endoplasmic reticulum stress, links sensing of metabolic stress to activation of pro-inflammatory cascades. Besides, JAK-STAT is also involved in DW healing by mediating the action of various innate immune effectors. Finally, we discuss the great potential of targeting these innate immune pathways and reprogramming innate immune cell phenotypes in DW therapy.

Grass carp (Ctenopharyngodon idella) TNK1 modulates JAK-STAT signaling through phosphorylating STAT1

TNK1 (thirty-eight-negative kinase 1) belongs to the ACK (Activated Cdc42 Kinases) family of intracellular non-receptor tyrosine kinases that usually acts as an important regulator in cytokine receptor-mediated intracellular signal transduction pathways. JAK-STAT signal pathway acts as a key point in cellular proliferation, differentiation and immunomodulatory. Mammalian TNK1 is involved in antiviral immunity and activation of growth factors. However, TNK1 has rarely been studied in fish. To evaluate the role of fish TNK1 in JAK-STAT pathway, we cloned the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) TNK1 (CiTNK1). CiTNK1 protein consists of N-terminal Tyrkc (tyrosine kinase) domain, C-terminal SH3 (Src homology 3) domain and Pro-rich domain. Phylogenetic analysis showed that CiTNK1 has a closer relationship with Danio rerio TNK1. The expression and phosphorylation of CiTNK1 in grass carp tissues and cells was increased under poly(I:C) stimulation. Subcellular localization and co-immunoprecipitation indicated that CiTNK1 is targeted in the cytoplasm and interacts with grass carp STAT1 (CiSTAT1). Co-transfection of CiTNK1 and CiSTAT1 into cells facilitated the expression of IFN I. This is because that the presence of CiTNK1 enhanced the phosphorylation of CiSTAT1 and causes activation of CiSTAT1. Our results revealed that TNK1 can potentiate the phosphorylation of STAT1 and then regulates JAK-STAT pathway to trigger IFN I expression in fish.

Insights on Molecular Mechanisms of Ovarian Development in Decapod Crustacea: Focus on Vitellogenesis-Stimulating Factors and Pathways.

Vitellogenesis in crustaceans is an energy-consuming process. Though the underlying mechanisms of ovarian maturation in decapod Crustacea are still unclear, evidence indicates the process to be regulated by antagonistically-acting inhibitory and stimulating factors specifically originating from X-organ/sinus gland (XO/SG) complex. Among the reported neuromediators, neuropeptides belonging to the crustacean hyperglycemic hormone (CHH)-family have been studied extensively. The structure and dynamics of inhibitory action of vitellogenesis-inhibiting hormone (VIH) on vitellogenesis have been demonstrated in several species. Similarly, the stimulatory effects of other neuropeptides of the CHH-family on crustacean vitellogenesis have also been validated. Advancement in transcriptomic sequencing and comparative genome analysis has led to the discovery of a large number of neuromediators, peptides, and putative peptide receptors having pleiotropic and novel functions in decapod reproduction. Furthermore, differing research strategies have indicated that neurotransmitters and steroid hormones play an integrative role by stimulating neuropeptide secretion, thus demonstrating the complex intertwining of regulatory factors in reproduction. However, the molecular mechanisms by which the combinatorial effect of eyestalk hormones, neuromediators and other factors coordinate to regulate ovarian maturation remain elusive. These multifunctional substances are speculated to control ovarian maturation possibly via the autocrine/paracrine pathway by acting directly on the gonads or by indirectly exerting their stimulatory effects by triggering the release of a putative gonad stimulating factor from the thoracic ganglion. Acting through receptors, they possibly affect levels of cyclic nucleotides (cAMP and cGMP) and Ca2+ in target tissues leading to the regulation of vitellogenesis. The “stimulatory paradox” effect of eyestalk ablation on ovarian maturation continues to be exploited in commercial aquaculture operations, and is outweighed by the detrimental physiological effects of this procedure. In this regard, the development of efficient alternatives to eyestalk ablation based on scientific knowledge is a necessity. In this article, we focus principally on the signaling pathways of positive neuromediators and other factors regulating crustacean reproduction, providing an overview of their proposed receptor-mediated stimulatory mechanisms, intracellular signaling, and probable interaction with other hormonal signals. Finally, we provide insight into future research directions on crustacean reproduction as well as potential applications of such research to aquaculture technology development.

Pulmonary adverse events of small molecule JAK inhibitors in autoimmune disease: systematic review and meta-analysis.

Small molecule tyrosine kinase inhibitors [smTKI, comprising mostly of Janus kinase (JAK) and to a lesser extent, spleen tyrosine kinase (SyK) inhibitors] modulate the cytokine receptor-mediated intracellular signal cascade, and are an effective treatment for autoimmune diseases and malignancies. As smTKI are novel, long-term safety is uncertain. Due to increasing use, characterization of their true adverse event profile is critical. We performed a systematic review and meta-analysis of all published trial data on the pulmonary and serious adverse effects of smTKIs in autoimmune disease. EMBASE, MEDLINE, CENTRAL and Pneumotox databases were searched up to April 2019 for randomized controlled trials, observational studies and post marketing surveillance, comparing any smTKI with placebo or another therapy, or as monotherapy at different doses. Primary outcomes comprised of any respiratory complications including upper and lower respiratory tract infections (URTI, LRTI), influenza, pneumonia, opportunistic respiratory infections, drug-induced interstitial lung disease, pulmonary embolism and lung neoplasm. We identified 4667 citations for screening, and selected 319 studies for full text review. Seventy-nine studies were analysed, including 47 randomized controlled trials, 25 observational studies and seven post-marketing surveillance studies, comprising 159652 participants. There were significantly increased risks of URTI [risk difference (RD) 0.03; 95% CI: 0.01, 0.05; P = 0.00; 36 studies, 14724 participants], LRTI (RD 0.01; 95% CI: 0.00, 0.02; P = 0.02; 24 studies, 12302 participants), influenza (RD 0.01; 95% CI: 0.00, 0.01; P = 0.04; 22 studies, 10684 participants), and pneumonia (RD 0.00; 95% CI: 0.00, 0.01; P = 0.02; 33 studies, 15511 participants). No increased risk was found for other respiratory complications, including pulmonary embolism. SmTKI increases the risk of non-opportunistic respiratory infections compared with placebo. The risk of any serious pulmonary adverse events is low.

Pulmonary Adverse Events of Small Molecule Tyrosine Kinase Inhibitors: Meta-Analysis and Systematic Review

Background Small molecule tyrosine kinase inhibitors (smTKI) are signalling molecules, which modulate the cytokine receptor-mediated intracellular signal cascade, and is an effective treatment for several autoimmune diseases and malignancies. As smTKI are novel, long-term safety is uncertain. Commonly reported side effects include infections, typically pulmonary and herpes zoster. As published data mainly examines tofacitinib trials, the class effect of smTKI is unknown. Methods We performed a meta-analysis of all published trial data on the pulmonary adverse effects of smTKIs. EMBASE, MEDLINE, and CENTRAL databases were searched up to September 2018 for randomized controlled trials, observational studies, case reports, and post marketing surveillance, comparing any smTKI with placebo or another therapy, or as monotherapy at different doses. Primary outcomes comprised of any respiratory complications including upper and lower respiratory tract infections, influenza, opportunistic respiratory infections, pneumonia, interstitial pneumonitis, pulmonary embolism, and lung neoplasm. Findings We identified 2253 citations for screening, and selected 150 for full text review. Seventy-five studies were analyzed, including 49 RCTs, 16 observational studies, three case reports, and seven post-marketing surveillance studies, comprising 153,942 participants. Median duration of RCTs was 24 weeks. The overall risk of any pulmonary adverse event with smTKI is low across RCTs and observational studies. There was significantly increased risk of developing upper respiratory tract infections (URTI) (RD 0·03, 95% CI 0·02 to 0·04, p<0·00001, 36 trials, 16419 participants), pneumonia (RD 0·0032; 95% CI 0·0003 to 0·0060, p=0·03, 31 trials, 15001 participants) and influenza (RD 0·0078; 95% CI 0·0031, 0·0126; p=0·001, 5 trials, 16395 participants) after smTKI exposure. No significantly increased risk was found for other respiratory complications. Interpretation SmTKI increases risk of URTI, pneumonia and influenza compared with placebo or conventional agents. The risk of any pulmonary adverse event with smTKI is low. Funding Nil

Decoding communication patterns of the innate immune system by quantitative proteomics.

The innate immune system is a collective network of cell types involved in cell recruitment and activation using a robust and refined communication system. Engagement of receptor-mediated intracellular signaling initiates communication cascades by conveying information about the host cell status to surrounding cells for surveillance and protection. Comprehensive profiling of innate immune cells is challenging due to low cell numbers, high dynamic range of the cellular proteome, low abundance of secreted proteins, and the release of degradative enzymes (e.g., proteases). However, recent advances in mass spectrometry-based proteomics provides the capability to overcome these limitations through profiling the dynamics of cellular processes, signaling cascades, post-translational modifications, and interaction networks. Moreover, integration of technologies and molecular datasets provide a holistic view of a complex and intricate network of communications underscoring host defense and tissue homeostasis mechanisms. In this Review, we explore the diverse applications of mass spectrometry-based proteomics in innate immunity to define communication patterns of the innate immune cells during health and disease. We also provide a technical overview of mass spectrometry-based proteomic workflows, with a focus on bottom-up approaches, and we present the emerging role of proteomics in immune-based drug discovery while providing a perspective on new applications in the future.

Cardiac molecular imaging to track left ventricular remodeling in heart failure

Cardiac left ventricular (LV) remodeling is the final common pathway of most primary cardiovascular diseases that manifest clinically as heart failure (HF). The more advanced the systolic HF and LV dysfunction, the worse the prognosis. The knowledge of the molecular, cellular, and neurohormonal mechanisms that lead to myocardial dysfunction and symptomatic HF has expanded rapidly and has allowed sophisticated approaches to understanding and management of the disease. New therapeutic targets for pharmacologic intervention in HF have also been identified through discovery of novel cellular and molecular components of membrane-bound receptor-mediated intracellular signal transduction cascades. Despite all advances, however, the prognosis of systolic HF has remained poor in general. This is, at least in part, related to the (1) relatively late institution of treatment due to reliance on gross functional and structural abnormalities that define the “heart failure phenotype” clinically; (2) remarkable genetic-based interindividual variations in the contribution of each of the many molecular components of cardiac remodeling; and (3) inability to monitor the activity of individual pathways to cardiac remodeling in order to estimate the potential benefits of pharmacologic agents, monitor the need for dose titration, and minimize side effects. Imaging of the recognized ultrastructural components of cardiac remodeling can allow redefinition of heart failure based on its “molecular phenotype,” and provide a guide to implementation of “personalized” and “evidence-based” evaluation, treatment, and longitudinal monitoring of the disease beyond what is currently available through randomized controlled clinical trials.

γ-Secretase Activity Is Required for Regulated Intramembrane Proteolysis of Tumor Necrosis Factor (TNF) Receptor 1 and TNF-mediated Pro-apoptotic Signaling

The γ-secretase protease and associated regulated intramembrane proteolysis play an important role in controlling receptor-mediated intracellular signaling events, which have a central role in Alzheimer disease, cancer progression, and immune surveillance. An increasing number of γ-secretase substrates have a role in cytokine signaling, including the IL-6 receptor, IL-1 receptor type I, and IL-1 receptor type II. In this study, we show that following TNF-converting enzyme-mediated ectodomain shedding of TNF type I receptor (TNFR1), the membrane-bound TNFR1 C-terminal fragment is subsequently cleaved by γ-secretase to generate a cytosolic TNFR1 intracellular domain. We also show that clathrin-mediated internalization of TNFR1 C-terminal fragment is a prerequisite for efficient γ-secretase cleavage of TNFR1. Furthermore, using in vitro and in vivo model systems, we show that in the absence of presenilin expression and γ-secretase activity, TNF-mediated JNK activation was prevented, assembly of the TNFR1 pro-apoptotic complex II was reduced, and TNF-induced apoptosis was inhibited. These observations demonstrate that TNFR1 is a γ-secretase substrate and suggest that γ-secretase cleavage of TNFR1 represents a new layer of regulation that links the presenilins and the γ-secretase protease to pro-inflammatory cytokine signaling.

The small leucine-rich repeat secreted protein Asporin induces eyes in Xenopus embryos through the IGF signalling pathway.

Small leucine-rich repeat proteoglycan (SLRP) family proteins play important roles in a number of biological events. Here, we demonstrate that the SLRP family member Asporin (ASPN) plays a crucial role in the early stages of eye development in Xenopus embryos. During embryogenesis, ASPN is broadly expressed in the neuroectoderm of the embryo. Overexpression of ASPN causes the induction of ectopic eyes. By contrast, blocking ASPN function with a morpholino oligonucleotide (ASPN-MO) inhibits eye formation, indicating that ASPN is an essential factor for eye development. Detailed molecular analyses revealed that ASPN interacts with insulin growth factor receptor (IGFR) and is essential for activating the IGF receptor-mediated intracellular signalling pathway. Moreover, ASPN perturbed the Wnt, BMP and Activin signalling pathways, suggesting that ASPN thereby creates a favourable environment in which the IGF signal can dominate. ASPN is thus a novel secreted molecule essential for eye induction through the coordination of multiple signalling pathways.

Integrins Influence the Size and Dynamics of Signaling Microclusters in a Pyk2-dependent Manner

Integrin engagement on lymphocytes initiates "outside-in" signaling that is required for cytoskeleton remodeling and the formation of the synaptic interface. However, the mechanism by which the "outside-in" signal contributes to receptor-mediated intracellular signaling that regulates the kinetics of granule delivery and efficiency of cytolytic activity is not well understood. We have found that variations in ICAM-1 expression on tumor cells influence killing kinetics of these cells by CD16.NK-92 cytolytic effectors suggesting that changes in integrin ligation on the effector cells regulate the kinetics of cytolytic activity by the effector cells. To understand how variations of the integrin receptor ligation may alter cytolytic activity of CD16.NK-92 cells, we analyzed molecular events at the contact area of these cells exposed to planar lipid bilayers that display integrin ligands at different densities and activating CD16-specific antibodies. Changes in the extent of integrin ligation on CD16.NK-92 cells at the cell/bilayer interface revealed that the integrin signal influences the size and the dynamics of activating receptor microclusters in a Pyk2-dependent manner. Integrin-mediated changes of the intracellular signaling significantly affected the kinetics of degranulation of CD16.NK-92 cells providing evidence that integrins regulate the rate of target cell destruction in antibody-dependent cell cytotoxicity (ADCC).

EphrinA2 receptor (EphA2) is an invasion and intracellular signaling receptor for Chlamydia trachomatis.

The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance.

Generation and Analysis of a Novel Mouse Model for Chronic Myelomonocytic Leukemia (CMML) with Acquired Expression of c-CBLQ367P

Myelodysplastic syndromes (MDS) are disorders originated from hematopoietic stem cells (HSCs), which are characterized by ineffective hematopoiesis, dysplasia mainly in the myeloid lineage, and high progression ratio to acute myeloid leukemia (AML). Recently, we identified mutations of the c-CBL (Casitas B cell lymphoma, a cellular homologue of v-CBL) gene in patients with MDS and myeloproliferative neoplasms (MPN). The mutations are detected in about 8% of the patients with the highest frequency in chronic myelomonocytic leukemia (CMML) cases with acquired uniparental disomy (aUPD) at 11q. c-CBL encodes a RING finger-based E3 ubiquitin ligase that negatively regulates receptor-mediated intracellular signaling. c-CBL is highly expressed in HSCs, strongly suggesting that it functions as a fine regulator of hematopoietic homeostasis. In fact, c-CBL knockout (KO) mice showed a myeloproliferative phenotype, owing to the hyper-responsiveness of HSCs to cytokine stimulation and subsequent augmented hematopoietic progenitor pool. In addition, c-CBL knockin (KI) mice harboring a mutation in the RING finger domain in one allele and a null mutation in the other allele exhibit an MPD-like disease and eventually progress to AML. These findings indicate that dysfunction of c-CBL perturbs normal hematopoietic development and contributes to hematopoietic abnormalities, but the precise leukemogenic mechanism(s) remains elusive. To gain insights into this issue and to create a novel animal model for mutated c-CBL-harboring leukemia, we generated conditional knock-in (cKI) mice that express wild-type c-CBL at steady state and inducibly express c-CBLQ367P, which was identified in patients with chronic myelomonocytic leukemia (CMML). After induced expression of c-CBLQ367P, the cKI mice exhibited a rapid and sustained increase in myelomonocytic cells with dysplasia in the peripheral blood and splenic enlargement with proliferation of myeloid cells, which closely resemble to the phenotype of human CMML. The bone marrow (BM) was hypercellular with predominance of myeloid cells, and increased number of HSC subpopulations and early myeloid progenitors were observed. In addition, phosphorylation of AKT, STAT3 and STAT5 was detected in long-term hematopoietic stem cells (LT-HSCs) of c-CBLQ367P cKI mice, indicating that PI3K/AKT and JAK/STAT signaling pathways are activated in c-CBLQ367P LT-HSCs. Moreover, competitive repopulation assays revealed that mice transplanted with c-CBLQ367P LT-HSCs showed significantly higher donor-derived chimerism than those transplanted with control LT-HSCs and displayed expansion of myelomonocytic cells as observed in c-CBLQ367P cKI mice, indicating that c-CBLQ367P conferred a proliferative advantage to LT-HSCs and that the phenotypes observed in c-CBLQ367P cKI mice were hematopoietic cell-intrinsic. CMML is known to progress to AML, possibly with additional genetic aberrations. To investigate the mechanism(s) underlying the disease evolution, we performed retroviral insertional mutagenesis using MOL4070A, a derivative of Moloney murine leukemia virus capable of inducing myeloid diseases. Almost all MOL4070A-infected c-CBLQ367P cKI mice developed AML, while no disease was observed in virus-injected control mice. Inverse PCR method identified Evi1 gene as a common integration site in the diseased mice and high Evi1 expression was detected in Evi1-integrated tumors. Mice transplanted with Evi1-transduced c-CBLQ367P cKI c-kit-positive BM cells developed AML at a high frequency and in a shortened period as compared to those transplanted with Evi1-transduced control cells. Taken together, we demonstrated that acquired expression of c-CBLQ367P plays a causative role in the development of CMML by activating PI3K/AKT and JAK/STAT pathways in HSCs and found that Evi1 overexpression cooperates with c-CBLQ367P to develop AML. Our mouse model provides a powerful tool for understanding of the pathogenesis of CMML and for developing novel therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.