Cytokine Receptor Signal Transduction Research Articles

JAK2/STAT5 inhibition protects mice from experimental autoimmune encephalomyelitis by modulating T cell polarization

Multiple sclerosis (MS) has been considered as a T cell-mediated autoimmune disease. However, the signaling pathways regulating effector T cells in MS have yet to be elucidated. Janus kinase 2 (JAK2) plays a crucial role in hematopoietic/immune cytokine receptor signal transduction. Here, we tested the mechanistic regulation of JAK2 and the therapeutic potential of pharmacological JAK2 inhibition in MS. Both inducible whole-body JAK2 knockout and T cell-specific JAK2 knockout completely prevented the onset of experimental autoimmune encephalomyelitis (EAE), a widely used MS animal model. Mice with JAK2 deficiency in T cells exhibited minimal demyelination and minimal CD45+ leukocyte infiltration in the spinal cord, accompanied by a remarkable reduction of T helper cell type 1 (TH1) and type 17 (TH17) in the draining lymph nodes and spinal cord. In vitro experiments showed that disruption of JAK2 markedly suppressed TH1 differentiation and IFNγ production. The phosphorylation of signal transducer and activator of transcription 5 (STAT5) was reduced in JAK2 deficient T cells, while STAT5 overexpression significantly increased TH1 and IFNγ production in STAT5 transgenic mice. Consistent with these results, JAK1/2 inhibitor baricitinib or selective JAK2 inhibitor fedratinib attenuated the frequencies of TH1 as well as TH17 in the draining lymph nodes and alleviated the EAE disease activity in mice. Our findings suggest that overactive JAK2 signaling in T lymphocytes is the culprit in EAE, which may serve as a potent therapeutic target for autoimmune disease.

Pharmacology of Janus kinase inhibitors

Modern dermatotherapy is dominated by the development of various biologicals and small molecules. Janus kinase inhibitors (JAKi) form anovel class of small molecular synthetic compounds inhibiting the intracellular signal transduction of cytokine receptors. Cytokines are key mediators in the pathophysiology of numerous inflammatory skin diseases. Many cytokines use so-called type I and II cytokine receptors, which associate with the Janus kinases JAK1, JAK2, JAK3 or TYK2. JAKi are under clinical investigation for inflammatory skin disease, specifically in phase3 trials for psoriasis or atopic dermatitis. Since JAKi are tested in oral as well as in topical formulations, they could become very popular in dermatotherapy. The mechanisms of JAKi, their selectivity, preliminary efficacy data, and their safety profile are discussed in this article.

SAT-091 Metabolic Effects of JAK1/2 Inhibition in Patients with Myeloproliferative Neoplasms

Background: Ruxolitinib is a JAK1/2 inhibitor that is FDA approved to treat certain myeloproliferative neoplasms (MPNs), including myelofibrosis (MF) and polycythemia vera (PV). The JAK family kinases are targeted in cancers due to their role in cytokine receptor signal transduction, and are also key intermediates in growth hormone, leptin and prolactin receptor signaling. Clinical trials have reported that ruxolitinib treatment was associated with weight gain, but other metabolic consequences remain unknown. Aim: We aimed to determine the metabolic consequences of JAK1/2 inhibition with ruxolitinib in patients with MPN in the clinical setting. Methods: IRB approval was obtained, and we identified patients from an electronic medical record (EMR) based database who began treatment with ruxolitinib for MPN from December 2011 to December 2016. Data collected from EMRs included age, gender, weight, height, systolic and diastolic blood pressure (SBP, DBP), medications, co-morbidities, random serum glucose, and lipid profile. We compared parameters from baseline (prior to initiating ruxolitinib) to 72±8 weeks after starting treatment. Baseline and 72 week data were compared using the Wilcoxon signed rank test. Results: 129 patients were initially identified, of which 71 had data available for weight, and at least one other metabolic parameter of interest at baseline and 72 weeks. Patient characteristics were as follows: 49.3% male; mean age at initiation of treatment was 64.6±SD11.1 years; indications for treatment were MF (77%), PV(17%), other MPNs (6%). Mean baseline weight was 73.6±16.9kg, and was 78.3±8.9kg at 72 weeks (n=71, p<0.001). Mean body mass index (BMI) at baseline was 25.6±4.6kg/m2, and 27.3±5.4kg/m2 at 72 weeks (n=68, p<0.001).The BMI (kg/m2) distribution for underweight (<18.5), normal (18.5-24.9), overweight (25.0-29.9), and obese (>30) at baseline were: 3%, 43%, 41%, and 13%, respectively; at 72 weeks it was 1%, 40%, 34%, and 28%, respectively (p=0.002). At 72 weeks,19% moved up a BMI class from baseline. Next, we investigated the effect of ruxolitinib treatment on BP and glucose concentrations. SBP was 124±15 mmHg at baseline and 129±18 mmHg at 72 weeks, (p=0.042, n=71). DBP was not different between baseline and 72 weeks. Glucose at baseline was 99±28mg/dL and 101±29mg/dL at 72 weeks (p=0.015, n=59). There was no change in the percent of patients with hyperglycemia (glucose ≥200mg/dL) or on treatment for diabetes at baseline (13.6%) and 72 weeks (13.6%). An insufficient number of patients had lipid data available for analysis. Conclusions: Systemic JAK1/2 inhibition was associated with weight gain, the development of obesity, and increased SBP in this cohort of patients. As pharmacological JAK1 and 2 inhibitors are developed and more widely used, it is important to gain a greater understanding of their long-term metabolic consequences.

E3 Ubiquitin Ligase c-cbl Inhibits Microglia Activation After Chronic Constriction Injury.

E3 ubiquitin ligase c-Caritas B cell lymphoma (c-cbl) is associated with negative regulation of receptor tyrosine kinases, signal transduction of antigens and cytokine receptors, and immune response. However, the expression and function of c-cbl in the regulation of neuropathic pain after chronic constriction injury (CCI) are unknown. In rat CCI model, c-cbl inhibited the activation of spinal cord microglia and the release of pro-inflammatory factors including tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β) and interleukin 6 (IL-6), which alleviated mechanical and heat pain through down-regulating extracellular signal-regulated kinase (ERK) pathway. Additionally, exogenous TNF-α inhibited c-cbl protein level vice versa. In the primary microglia transfected with c-cbl siRNA, when treated with TNF-α or TNF-α inhibitor, the corresponding secretion of IL-1β and IL-6 did not change. In summary, CCI down-regulated c-cbl expression and induced the activation of microglia, then activated microglia released inflammatory factors via ERK signaling to cause pain. Our data might supply a novel molecular target for the therapy of CCI-induced neuropathic pain.

A proline deletion in IFNAR1 impairs IFN-signaling and underlies increased resistance to tuberculosis in humans

Type I interferons (IFN), best known for their anti-viral functions, have been shown to impair host resistance to intracellular bacteria in mice. However, the precise role of type I IFN signaling in bacterial infection in humans is unclear. Here, we show that genetic variation in the human IFNAR1 gene is associated with decreased susceptibility to tuberculosis and an increased risk of viral hepatitis in Chinese populations. Receptor mutagenesis and cell signaling studies establish that the IFNAR1 mutation corresponding to a proline deletion in the hinge region of the membrane-proximal domain of IFNAR1 decreases the binding affinity of IFNAR1 to IFN-β, impeding type I IFN signaling. Our findings suggest that IFNAR1 signaling underlies an increased risk of tuberculosis in humans and reveals a function for the IFNAR1 inter-domain region in cytokine–cytokine receptor interaction and signal transduction.

Cbl-mediated K63-linked ubiquitination of JAK2 enhances JAK2 phosphorylation and signal transduction

JAK2 activation is crucial for cytokine receptor signal transduction and leukemogenesis. However, the underlying processes that lead to full activation of JAK2 are unclear. Here, we report a positive role for ubiquitination of JAK2 during GM-CSF-induced activation. Upon GM-CSF stimulation, JAK2 ubiquitination is significantly enhanced through K63-linked poly-ubiquitination. Studies employing both knockout and overexpression of Cbl, an E3 ubiquitin ligase, led to the conclusion that Cbl specifically promotes JAK2 ubiquitination, and this was further confirmed in vitro using a Cbl ubiquitination assay. Moreover, following GM-CSF stimulation, the levels of phospho-JAK2 and -STAT5 and a STAT5 luciferase reporter assay were all reduced in Cbl knockout cells and this effect could be rescued by Cbl expression. Mechanistically, Cbl can interact with, and ubiquitinate JAK2 FERM and kinase domains via the Cbl TKB domain. Using lysine-to-arginine site-directed mutagenesis, K970 in the kinase domain of JAK2 was identified as the ubiquitination site important for promoting full JAK2 activation by Cbl via K63-conjugated poly-ubiquitination. Our study suggests that GM-CSF-induced JAK2 activation is enhanced by Cbl-mediated ubiquitination of JAK2. Targeting ubiquitination of JAK2 might offer a novel therapeutic strategy against JAK2-mediated disorders.

Long-term Benefit of PD-L1 Blockade in Lung Cancer Associated with JAK3 Activation.

PD-1 immune checkpoint blockade occasionally results in durable clinical responses in advanced metastatic cancers. However, mechanism-based predictors of response to this immunotherapy remain incompletely characterized. We performed comprehensive genomic profiling on a tumor and germline sample from a patient with refractory lung adenocarcinoma who achieved marked long-term clinical benefit from anti-PD-L1 therapy. We discovered activating somatic and germline amino acid variants in JAK3 that promoted PD-L1 induction in lung cancer cells and in the tumor immune microenvironment. These findings suggest that genomic alterations that deregulate cytokine receptor signal transduction could contribute to PD-L1 activation and engagement of the PD-1 immune checkpoint in lung cancer.

SUMOylation is a regulator of the translocation of Jak2 between nucleus and cytosol

Jak2 (Janus kinase 2) initiates the signal transduction of many cytokine receptors. We discovered that Jak2 is SUMOylated on multiple lysine residues by SUMO2/3 (small ubiquitin-related modifier 2/3) chains. Analysis of Jak2 mutants revealed that Jak2 SUMOylation depends on the presence of an active catalytic site. We used the GH (growth hormone) receptor to study the physiological relevance of Jak2 SUMOylation. Both GH stimulation and several other environmental stressors increased Jak2 SUMOylation. Cell fractionation showed that SUMOylated Jak2 is mainly present in the nucleus. The constitutively active V617F Jak2 mutant, implicated in myeloproliferative diseases, was highly SUMOylated in the absence of stimuli. These data provide evidence that Jak2 SUMOylation controls Jak2 shuttling between cytoplasm and nucleus.

Jak2 Is a Negative Regulator of Ubiquitin-Dependent Endocytosis of the Growth Hormone Receptor

BackgroundLength and intensity of signal transduction via cytokine receptors is precisely regulated. Degradation of certain cytokine receptors is mediated by the ubiquitin ligase SCF(βTrCP). In several instances, Janus kinase (Jak) family members can stabilise their cognate cytokine receptors at the cell surface.Principal FindingsIn this study we show in Hek293 cells that Jak2 binding to the growth hormone receptor prevents endocytosis in a non-catalytic manner. Following receptor activation, the detachment of phosphorylated Jak2 induces down-regulation of the growth hormone receptor by SCF(βTrCP). Using γ2A human fibroblast cells we show that both growth hormone-induced and constitutive growth hormone receptor endocytosis depend on the same factors, strongly suggesting that the modes of endocytosis are identical. Different Jak2 RNA levels in HepG2, IM9 and Hek293 cells indicate the importance of cellular concentration on growth hormone receptor function. Both Jak2 and βTrCP bind to neighbouring linear motifs in the growth hormone receptor tail without the requirement of modifications, indicating that growth hormone sensitivity is regulated by the cellular level of non-committed Jak2.Conclusions/SignificanceAs signal transduction of many cytokine receptors depends on Jak2, the study suggests an integrative role of Jak2 in cytokine responses based on its enzyme activity as well as its stabilising properties towards the receptors.

WP-1130 Induces Apoptosis in Multiple Myeloma Cells by Inactivation of Jak2 and Syk Tyrosine Kinases Via a Novel Mechanism of Selective Protein Trafficking

Multiple myeloma is a plasma cell malignancy affecting more than 50,000 people in the United States. The disease remains incurable despite significant recent advances in treatment, including the immunomodulatory agents, thalidomide and lenalidomide, and the proteasome inhibitor, bortezomib. Thus the search for additional agents is warranted. We have previously shown that WP1130, a tyrphostin-like compound, has activity in a number of hematological malignancies including CML, mantle cell lymphoma and multiple myeloma. In this study, we investigated the activity and mechanism of action of WP1130 on multiple myeloma cells and compared these activities to bortezomib. Both WP1130 and bortezomib demonstrated significant cytotoxicity in MM1S cells with IC50s of 1 mM and 2 nM, respectively and both compounds induced similar levels of apoptosis (67% vs 73% cell death with WP1130 and bortezomib, respectively). Both WP1130 and bortezomib increased cellular ubiquitination levels in distinct cellular compartments but we were unable to detect 20S proteasome inhibition by WP1130. The effect of WP1130 on signaling modules in MM cells was also investigated. WP1130 altered the subcellular distribution of a subset of tyrosine kinases, including Jak2 and Syk which were irreversibly displaced from the cytoplasm to detergent insoluble complexes within the cell. Although Jak2 remained stable in this fraction, Syk was subject to proteolysis and destruction in this compartment. WP1130-induced translocation of these kinases through association with the cytoskeletal network and was associated with loss of cytokine and B-cell receptor signal transduction. WP1130 did not alter the cellular distribution of other tyrosine kinase or phosphoproteins including Lyn, STAT3, STAT5, PI3K, AKT, ERK, suggesting some selective protein translocation and signal modulation by WP1130. These results support a novel mechanism of protein trafficking, selective kinase destruction and apoptosis by WP1130 in MM cells.

Improvement of Photoaffinity SPR Imaging Platform and Determination of the Binding Site of p62/SQSTM1 to p38 MAP Kinase

p38 mitogen-activated protein kinase (MAPK) is a member of the serine/threonine kinases and is activated in response to stress stimuli, such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock. We revealed in a previous report that p62/SQSTM1, known to participate in proteasomal or autophagosomal protein degradation and cytokine receptor signal transduction pathways, binds to p38 to regulate specifically. Herein, we describe the improvement of the photoaffinity-thiol linker of our SPR imaging platform, which enabled us to determine the binding site of p62 to p38. SPR imaging experiments using a new photoaffinity linker 2 to immobilize the peptides derived from p62 on gold substrate indicate that the domain comprising amino acids 164-190 of p62 binds to p38 directly. These SPR analysis data and empirical biologic data reveal that the binding site of p62 to p38 is the domain corresponding to 173-182.

Acetylation-Dependent Signal Transduction for Type I Interferon Receptor

Cytokine-activated receptors initiate intracellular signaling by recruiting protein kinases that phosphorylate the receptors on tyrosine residues, thus enabling docking of SH2 domain-bearing activating factors. Here we report that in response to type 1 interferon (IFNalpha), IFNalpha receptors recruit cytoplasmic CREB-binding protein (CBP). By binding to IFNalphaR2 within the region where two adjacent proline boxes bear phospho-Ser364 and phospho-Ser384, CBP acetylates IFNalphaR2 on Lys399, which in turn serves as the docking site for interferon regulatory factor 9 (IRF9). IRF9 interacts with the acetyl-Lys399 motif by means of its IRF homology2 (IH2) domain, leading to formation of the ISGF3 complex that includes IRF9, STAT1, and STAT2. All three components are acetylated by CBP. Remarkably, acetylation within the DNA-binding domain (DBD) of both IRF9 and STAT2 is critical for the ISGF3 complex activation and its associated antiviral gene regulation. These results have significant implications concerning the central role of acetylation in cytokine receptor signal transduction.

Ligation of CD28 Stimulates the Formation of a Multimeric Signaling Complex Involving Grb-2-Associated Binder 2 (Gab2), Src Homology Phosphatase-2, and Phosphatidylinositol 3-Kinase: Evidence That Negative Regulation of CD28 Signaling Requires the Gab2 Pleckstrin Homology Domain

Grb-2-associated binder (Gab)2 is a scaffolding adaptor protein that has been reported to promote growth factor and cytokine receptor signal transduction, but inhibit TCR-mediated signaling events. In this study, we show that ligation of CD28 by its natural ligand B7-1/CD80, induces tyrosine phosphorylation of Gab2 and its coassociation with Src homology phosphatase (SHP)-2 and class IA PI3K in Jurkat cells. Overexpression of wild-type Gab2 revealed a negative role in regulation of CD3/CD28 induction of the transcription factors NF-kappaB and AP-1. To characterize this inhibitory function further, we used Gab2 mutants unable to bind either PI3K or SHP-2 and a PH domain deletion mutant. Although PI3K has previously been implicated as necessary for Gab2-mediated inhibition of TCR signaling, Gab2 mutants defective in their ability to bind PI3K or SHP-2 retained their inhibitory function, whereas deletion of the PH domain ablated the inhibitory effect of Gab2. Together, these data demonstrate that CD28 stimulation of T cells is sufficient to induce an inhibitory multimeric signaling complex involving Gab2, SHP-2, and PI3K. Furthermore, the inhibitory capacity of Gab2 is strictly dependent upon the integrity of its PH domain, suggesting phosphoinositide-mediated membrane recruitment is important to Gab2 function in T cells.

Stat4-null non-obese diabetic mice: protection from diabetes and experimental allergic encephalomyelitis, but with concomitant epitope spread

There is much interest in therapeutic manipulation of cytokine responses in autoimmunity, yet studies in mouse models have sometimes produced conflicting findings as to the role of particular mediators in disease. Examples include the contradictory findings regarding susceptibility to experimental allergic encephalomyelitis (EAE) or diabetes in knockout mice for various individual Th1 or Th2 cytokines or their receptors. An alternative approach to the analysis of Th1 and Th2 mechanisms in these diseases is to investigate strains carrying a null mutation for molecules involved in cytokine receptor signal transduction, signal transducer and activator of transcription (Stat4) and Stat6. Stat4 is pivotal in Th1 polarization, being activated when IL-12 binds the IL-12R and leading to the production of IFNgamma. We here report disease susceptibility in non-obese diabetic mice carrying a Stat4-null mutation. Knockout mice were almost completely protected from diabetes, only rarely showing pancreatic peri-islet infiltrates. Furthermore, there was near complete protection from the induction of EAE by either of the two encephalitogenic myelin epitopes. Despite this protection, Stat4-null mice showed clear epitope spread compared with controls during myelin oligodendrocyte glycoprotein-induced EAE as judged by T cell proliferation, although this was not associated with a strong Th1 response to the initial or spread epitope and, furthermore, there was no evidence of a switch to Th2 cytokines.

New insights into leptin resistance by modifying cytokine receptor signal transduction

It has been widely accepted that leptin treatment of obese humans is restricted to those few subjects that suffer from congenital leptin deficiency (1). The peripheral administration of leptin to overweight patients with fat mass-adjusted normal serum leptin concentrations does not reduce body weight substantially (2). These observations suggest that leptin resistance in obese human beings is similar to insulin resistance in patients with type 2 diabetes mellitus. Several mechanisms contributing to leptin resistance have been identified so far. First, a saturable transporter allowing leptin to pass the blood –brain barrier was discovered. This mechanism impedes the hormone’s transfer to the hypothalamus once certain plasma concentrations are exceeded (3, 4). Secondly, negative regulators of leptin signalling such as the tyrosine phosphatases protein tyrosine phosphatase 1B and SH-containing protein tyrosine phosphatase (5–8) as well as the suppressors of cytokine signalling (Socs) (9) have been shown to contribute to leptin resistance. Since leptin indeed acts via a cytokine receptor, the latter group is of particular interest. Socs molecules inhibit the function of cytokines and of other proteins such as leptin by interfering with receptor-dependent JAKs or STATs (Fig. 1). In fact, peripheral leptin administration in mice has previously been shown to increase the hypothalamic concentration of Socs3 (10). This would suggest that increased serum concentrations of leptin in obese subjects might impede leptin function by interfering with receptor signalling through Socs proteins. At first glance it appears to be simple to test this hypothesis by watching the effect of leptin in a Socs3-deficient knock-out mouse model. However, homozygous Socs3 knock-out mice are stillborn due to placental defects (11). Therefore, it is of great interest that two recent articles eventually shed light on the role of Socs3, the suppressor of leptin function, using two different approaches. In the first report J S Flier’s group used a heterozygous Socs3 þ /2mouse model to demonstrate in this non-lethal animal model reduced Socs3 mRNA expression in the hypothalami (12). The second report used a Cre-loxP system to allow neural cellspecific specific Socs3 knock-out not interfering with the non-neuronal functions of the protein (13). They were able to demonstrate complete absence of Socs3 in the brain of their animals. Using these two different approaches the two papers achieve amazingly consistent findings: (i) Socs3 þ /2 animals as well as neural cell-specific knock-out mice do not gain significantly more weight or increase food intake compared with their normally fed littermates even when offered a high-fat diet. In contrast, wildtype mice developed a significant increase in food intake and body weight. This suggests that leptininduced suppression of appetite is enhanced in the Socs3-deficient animals and that Socs3 is a mediator of leptin resistance. (ii) While wild-type mice develop insulin resistance when exposed to high-fat diet this is not the case in the two different knock-out animals with reduction or absence of Socs3 in the brain. This could be demonstrated by normalisation of insulin secretion as well as a normalised response to glucose tolerance tests. (iii) With regard to the mechanism of action, in both studies the hypothalamic response to peripheral leptin administration was increased in the knock-out animals, suggesting a lesser degree of impairment of leptin receptor function. Specifically, STAT3 phosphorylation, being the most important mode of signal transduction of leptin, was increased in both the heterozygous Socs3 and the neural cellspecific Socs3 knock-out following leptin application. However, while the principal messages of both communications on Socs3 suppression are in line, some of the findings are distinct: Howard et al. (12) do not report alterations in the hypothalamic gene expression of the leptin receptor downstream peptides neuropeptide Y (NPY), proopiomelanocortin (POMC) and agoutirelated peptide (AgRP) with their heterozygous Socs3 knock-out between wild-type and knock-out mice after leptin infusion. This leads them to suggest that other downstream peptides might be of relevance for leptin action. In contrast, the complete Socs3 knock-out in brain tissue that is reached in experiments by Mori et al. (13) leads to an increase in hypothalamic POMC mRNA in contrast to wild-type animals 6 h after leptin infusion. This shows how important different approaches may be to elucidate the exact mechanisms of leptin action. H IG H L IG H T European Journal of Endocrinology (2005) 152 333–334 ISSN 0804-4643

Generation of a conditional knockout allele for the Janus kinase 2 (Jak2) gene in mice.

To study biologically relevant functions of the Janus kinase 2 (Jak2) in multiple cytokine and hormone receptor signal transduction pathways, we generated a conditional knockout (floxed) allele of this gene by placing loxP sites around the first coding exon of Jak2. Homozygous floxed animals developed normally and exhibited no phenotypic abnormalities. The conversion of the floxed allele into a null mutation was achieved by transmitting the targeted allele through the female germline of MMTV-Cre (line A) mice. Embryos that carry two Jak2 null alleles died around midgestation and exhibited impaired definitive erythropoiesis, which is a hallmark of Jak2 deficiency reported previously in conventional knockouts. This observation suggested that the Cre-mediated deletion of the first coding exon results in a true null mutation that is incapable of mediating signals through the erythropoietin receptor. Using mouse embryonic fibroblasts derived from Jak2 null embryos and their wildtype littermate controls, we demonstrated that Jak2-deficiency decouples growth hormone-receptor signaling from its downstream mediators, the signal transducer and activator of transcription (Stat) 5a and 5b.

Differentiation of acute and chronic myeloid leukemic blasts into the dendritic cell lineage: analysis of various differentiation-inducing signals.

Ex vivo differentiation of myeloid leukemic blasts into dendritic cells (DCs) holds significant promise for use as cellular vaccines, as they may present a constellation of endogenously expressed known and unknown leukemia antigens to the immune system. Although variety of stimuli can drive leukemia --> DC differentiation in vitro, these blast-derived DCs typically have aberrant characteristics compared with DCs generated from normal progenitors by the same stimuli. It is not clear whether this is due to underlying leukemogenic mechanisms (e.g., specific oncogenes), genetic defects, stage of maturation arrest, defects in cytokine receptor expression or signal transduction pathways, or whether different stimuli themselves induce qualitatively dissimilar DC differentiation. To assess what factors may contribute to aberrant leukemic blast --> DC differentiation, we have examined how the same leukemic blasts (AML and CML) respond to different DC differentiation signals--including extracellular (the cytokine combination GM-CSF + TNF-alpha + IL-4) and intracellular (the protein kinase C agonist PMA, the calcium ionophore A23187, and the combination of PMA plus A23187) stimuli. We have found that the same leukemic blasts will develop qualitatively different sets of DC characteristics in response to differing stimuli, although no stimuli consistently induced all of the characteristic DC features. There were no clear differences in the responses relative to specific oncogene expression or stage of maturation arrest (AML vs CML). Signal transduction agonists that bypassed membrane receptors/proximal signaling (in particular, the combination of PMA and A23187) consistently induced the greatest capability to activate T cells. Interestingly, this ability did not clearly correlate with expression of MHC/costimulatory ligands. Our findings suggest that signal transduction may play an important role in the aberrant DC differentiation of leukemic blasts, and demonstrate that direct activation of PKC together with intracellular calcium signaling may be an effective method for generating immunostimulatory leukemia-derived DCs.

The role of Gab family scaffolding adapter proteins in the signal transduction of cytokine and growth factor receptors.

The Grb2-associated binder (Gab) family adapter proteins are scaffolding adapter molecules that display sequence similarity with Drosophila DOS (daughter of sevenless), which is a substrate for the protein tyrosine phosphatase Corkscrew. Gab proteins contain a pleckstrin homology (PH) domain and binding sites for SH2 and SH3 domains. A number of studies in multiple systems have implicated Gab in signaling via many different types of receptors, such as growth factor, cytokine, and antigen receptors, and via oncoproteins. Recent studies of Gab1 and Gab2 knockout mice have clearly indicated an important role for Gabs in vivo. Gab1-deficient mice die as embryos with multiple defects in placental, heart, skin, and muscle development. Gab2-deficient mice are viable, but have a defect in the mast cell lineages and in allergic reactions. Given the apparently central role played by Gab signaling via many receptors, delineating the precise mechanism(s) of Gab-mediated signaling is critical to understanding how cytokines, growth factors, and oncoproteins mediate a variety of biological activities: cell growth, differentiation, survival and malignant transformation.

Fill a Gab(1) in Cardiac Hypertrophy Signaling

Cardiac hypertrophy represents one of the most central issues in heart failure, involving many aspects of changes in cardiac myocyte from cellular morphology to gene expression and contractile function.1,2 Although the process is at the beginning compensatory in response to hemodynamic load or myocardial injury, cardiac hypertrophy is often followed by pathological remodeling in myocardium and a transition to overt heart failure.3 A large number of extracellular ligands, their cognate receptors and corresponding intracellular signal transduction pathways have been implicated in the hypertrophic process from both in vitro and in vivo studies.4–13 Among them, cardiotrophin-1 (CT-1) was discovered as a potent activator of cardiac hypertrophy14 that shares extensive sequence and functional similarities with leukemia inhibitory factor (LIF) as members of interleukin-6 (IL-6)–related cytokine family.15 LIF/CT-1 bind to gp130/LIF cytokine receptor β heterodimer and activate downstream signaling pathways, including members of signal transducer and activator of transcription (STATs), mitogen-activated protein (MAP) kinases such as extracellular signal–regulated kinases (ERK1/2), and phosphatidylinositol 3-kinase (PI3K)/Akt.15,16 Studies have demonstrated that both LIF and CT-1 promote myocyte hypertrophy, survival, and a unique pattern of embryonic/fetal gene induction.17–22 LIF and CT-1 expression and gp130 signaling also correlated with mechanical stress in myocytes in culture23 and human and experimental models of heart failure.24–30 More significantly, targeted inactivation of gp130 in ventricular myocytes via Cre-loxP–mediated tissue-specific knockout resulted in rapid chamber dilation and a significant increase in myocyte apoptosis upon pressure overload.31 All these findings underscore the potential function of gp130 signaling in …

Flow cytometric analysis of cytokine receptor signal transduction

Cytokines are critical regulators of the development and maturation of hematopoietic cells. Signal transduction via cytokine receptors proceeds through activation of the JAK–STAT pathway to stimulate cell proliferation, differentiation and effector functions. Phosphorylation of intracellular STAT molecules by the receptor-associated JAK kinases is one of the very early events following cytokine stimulation. Western blot detection of tyrosine phosphorylated STAT molecules is widely used as a hallmark of cytokine receptor activation. However, this is not feasible when cells of interest are limiting, or represent a small fraction within a mixed population of different cell types. To circumvent this technical obstacle, we have developed techniques to detect phosphorylated STAT molecules in fixed cells by flow cytometry. The fixation and permeabilization protocols preserve the antigenicity of cell surface markers allowing us to distinguish distinct cell populations responding to cytokine stimulation. In this report, we demonstrate the use of this technique to rapidly and reliably identify, and quantify thymocyte subsets activated by interleukin-7. We envisage that this technique will find wide application in studying cytokine receptor signal transduction, particularly in identifying cytokine-dependent developmental checkpoints during hematopoiesis.