J. Luis Morales

Absence of Tec Family Kinases Interleukin-2 Inducible T cell Kinase (Itk) and Bruton's Tyrosine Kinase (Btk) Severely Impairs FcϵRI-dependent Mast Cell Responses

Mast cells are critical effector cells in the pathophysiology of allergic asthma and other IgE-mediated diseases. The Tec family of tyrosine kinases Itk and Btk serve as critical signal amplifiers downstream of antigen receptors. Although both kinases are expressed and activated in mast cells following FcϵRI stimulation, their individual contributions are not clear. To determine whether these kinases play unique and/or complementary roles in FcϵRI signaling and mast cell function, we generated Itk and Btk double knock-out mice. Analyses of these mice show decreased mast cell granularity and impaired passive systemic anaphylaxis responses. This impaired response is accompanied by a significant elevation in serum IgE in Itk/Btk double knock-out mice. In vitro analyses of bone marrow-derived mast cells (BMMCs) indicated that Itk/Btk double knock-out BMMCs are defective in degranulation and cytokine secretion responses downstream to FcϵRI activation. These responses were accompanied by a significant reduction in PLCγ2 phosphorylation and severely impaired calcium responses in these cells. This defect also results in altered NFAT1 nuclear localization in double knock-out BMMCs. Network analysis suggests that although they may share substrates, Itk plays both positive and negative roles, while Btk primarily plays a positive role in mast cell FcϵRI-induced cytokine secretion.

Structural Requirements for the Inhibition of Calcium Mobilization and Mast Cell Activation by the Pyrazole Derivative BTP2

Mast cells play a critical role in the development of the allergic response. Upon activation by allergens and IgE via the high affinity receptor for IgE (FcɛRI), these cells release histamine and other functional mediators that initiate and propagate immediate hypersensitivity reactions. Mast cells also secrete cytokines that can regulate immune activity. These processes are controlled, in whole or part, by increases in intracellular Ca(2+) induced by the FcɛRI. We show here that N-(4-(3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2), a pyrazole derivative, inhibits activation-induced Ca(2+) influx in the rat basophil cell line RBL-2H3 and in bone marrow-derived mast cells (BMMCs), without affecting global tyrosine phosphorylation of cellular proteins or phosphorylation of the mitogen-activated protein kinases Erk1/2, JNK and p38. BTP2 also inhibits activation-induced degranulation and secretion of interleukin (IL)-2, IL-3, IL-4, IL-6, IL-13, tumor necrosis factor (TNF)-α, and granulocyte macrophage-colony stimulating factor (GM-CSF) by BMMCs, which correlates with the inhibition of Nuclear Factor of Activated T cells (NFAT) translocation. In vivo, BTP2 inhibits antigen-induced histamine release. Structure-activity relationship analysis indicates that substitution at the C3 or C5 position of the pyrazole moiety on BTP2 (5-trifluoromethyl-3-methyl-pyrazole or 3-trifluoromethyl-5-methyl-pyrazole, respectively) affected its activity, with the trifluoromethyl group at the C3 position being critical to its activity. We conclude that BTP2 and related compounds may be potent modulators of mast cell responses and potentially useful for the treatment of symptoms of allergic inflammation.

Chemico-Genetic Identification of Drebrin as a Regulator of Calcium Responses

Store-operated calcium channels are plasma membrane Ca(2+) channels that are activated by depletion of intracellular Ca(2+) stores, resulting in an increase in intracellular Ca(2+) concentration, which is maintained for prolonged periods in some cell types. Increases in intracellular Ca(2+) concentration serve as signals that activate a number of cellular processes, however, little is known about the regulation of these channels. We have characterized the immuno-suppressant compound BTP, which blocks store-operated channel mediated calcium influx into cells. Using an affinity purification scheme to identify potential targets of BTP, we identified the actin reorganizing protein, drebrin, and demonstrated that loss of drebrin protein expression prevents store-operated channel mediated Ca(2+) entry, similar to BTP treatment. BTP also blocks actin rearrangements induced by drebrin. While actin cytoskeletal reorganization has been implicated in store-operated calcium channel regulation, little is known about actin-binding proteins that are involved in this process, or how actin regulates channel function. The identification of drebrin as a mediator of this process should provide new insight into the interaction between actin rearrangement and store-operated channel mediated calcium influx.

Estrogen Receptor Expression Is Required for Low-Dose Resveratrol-Mediated Repression of Aryl Hydrocarbon Receptor Activity

The putative cardioprotective and chemopreventive properties of the red wine phenolic resveratrol (RES) have made it the subject of a growing body of clinical and basic research. We have begun investigations focusing on the effects of RES on the activity of the aryl hydrocarbon receptor (AHR) complex. Our evidence suggests that RES is a potent repressor of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible gene transcription in estrogen receptor (ER)-positive human breast, lung, and colon cancer cell lines. RES activates the transcription of the ER target genes to the same degree as estradiol (E2) in human MCF-7 breast cancer cells. Unlike E2, which can only diminish TCDD-inducible CYP1A1 gene transcription by approximately 50%, RES can completely abrogate this response. Furthermore, 50% repression of TCDD-inducible transcription can be achieved with 100 nM RES, approximately 2.5 orders of magnitude lower than concentrations required for maximal inhibition, suggesting that multiple mechanisms are responsible for this effect. RES (100 nM) does not prevent ligand binding of a TCDD analog, nor does it prevent AHR from binding to its response element in the 5′-regulatory region of the CYP1A1 gene. Small inhibitory RNAs directed to ERα have demonstrated that RES-mediated repression of CYP1A1 depends on ERα. Whereas CYP1A1 protein levels in MCF-7 cells are refractory to the low-dose transcriptional effects of RES, a concomitant decrease in CYP1A1 protein levels is observed in Caco-2 cells. These results highlight a low-dose RES effect that could occur at nutritionally relevant exposures and are distinct from the high-dose effects often characterized.

Modeling Susceptibility versus Resistance in Allergic Airway Disease Reveals Regulation by Tec Kinase Itk

Murine models of allergic asthma have been used to understand the mechanisms of development and pathology in this disease. In addition, knockout mice have contributed significantly to our understanding of the roles of specific molecules and cytokines in these models. However, results can vary significantly depending on the mouse strain used in the model, and in particularly in understanding the effect of specific knockouts. For example, it can be equivocal as to whether specific gene knockouts affect the susceptibility of the mice to developing the disease, or lead to resistance. Here we used a house dust mite model of allergic airway inflammation to examine the response of two strains of mice (C57BL/6 and BALB/c) which differ in their responses in allergic airway inflammation. We demonstrate an algorithm that can facilitate the understanding of the behavior of these models with regards to susceptibility (to allergic airway inflammation) (Saai) or resistance (Raai) in this model. We verify that both C57BL/6 and BALB/c develop disease, but BALB/c mice have higher Saai for development. We then use this approach to show that the absence of the Tec family kinase Itk, which regulates the production of Th2 cytokines, leads to Raai in the C57BL/6 background, but decreases Saai on the BALB/c background. We suggest that the use of such approaches could clarify the behavior of various knockout mice in modeling allergic asthma.

Cutting Edge: Drebrin-Regulated Actin Dynamics Regulate IgE-Dependent Mast Cell Activation and Allergic Responses

Mast cells play critical roles in allergic responses. Calcium signaling controls the function of these cells, and a role for actin in regulating calcium influx into cells has been suggested. We have previously identified the actin reorganizing protein Drebrin as a target of the immunosuppressant 3,5-bistrifluoromethyl pyrazole, which inhibits calcium influx into cells. In this study, we show that Drebrin−/− mice exhibit reduced IgE-mediated histamine release and passive systemic anaphylaxis, and Drebrin−/− mast cells also exhibit defects in FcεRI-mediated degranulation. Drebrin−/− mast cells exhibit defects in actin cytoskeleton organization and calcium responses downstream of the FcεRI, and agents that relieve actin reorganization rescue mast cell FcεRI-induced degranulation. Our results indicate that Drebrin regulates the actin cytoskeleton and calcium responses in mast cells, thus regulating mast cell function in vivo.

Nonreceptor tyrosine kinases ITK and BTK negatively regulate mast cell proinflammatory responses to lipopolysaccharide

BACKGROUND Mast cells are indispensable for LPS-induced septic hypothermia, in which TNF-α plays an essential role to initiate septic responses. ITK and BTK regulate mast cell responses to allergens, but their roles in mast cell responses in LPS-induced sepsis are unclear. OBJECTIVE We sought to investigate the roles of ITK and BTK in mast cell responses during LPS-induced septic inflammation. METHODS Mice (genetically modified or bone marrow-derived mast cell-reconstituted Sash) were given LPS to induce septic hypothermia in the presence or absence of indicated inhibitors. Flow cytometry was used to determine LPS-induced cell influx and TNF-α production in peritoneal cells. Microarray was used for genomewide gene expression analysis on bone marrow-derived mast cells. Quantitative PCR and multiplex were used to determine transcribed and secreted proinflammatory cytokines. Microscopy and Western blotting were used to determine activation of signal transduction pathways. RESULTS The absence of ITK and BTK leads to exacerbation of LPS-induced septic hypothermia and neutrophil influx. Itk(-/-)Btk(-/-) mast cells exhibit hyperactive preformed and LPS-induced TNF-α production, and lead to more severe LPS-induced septic hypothermia when reconstituted into mast cell-deficient Sash mice. LPS-induced nuclear factor kappa B, Akt, and p38 activation is enhanced in Itk(-/-)Btk(-/-) mast cells, and blockage of phosphatidylinositol-4,5-bisphosphate 3-kinase, Akt, or p38 downstream mitogen-activated protein kinase interacting serine/threonine kinase 1 activation significantly suppresses TNF-α hyperproduction and attenuates septic hypothermia. CONCLUSIONS ITK and BTK regulate thermal homeostasis during septic response through mast cell function in mice. They share regulatory function downstream of Toll-like receptor 4/LPS in mast cells, through regulating the activation of canonical nuclear factor kappa B, phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt, and p38 signaling pathways.

The zinc-binding region of IL-2 inducible T cell kinase (Itk) is required for interaction with Gα13 and activation of serum response factor.

Tec family kinases play critical roles in the activation of immune cells. In particular, Itk is important for the activation of T cells via the T cell Receptor (TcR), however, molecules that cooperate with Itk to activate downstream targets remain little explored. Here we show that Itk interacts with the heterotrimeric G-protein α subunit Gα13 during TcR triggering. This interaction requires membrane localization of both partners, and is partially dependent on GDP- and GTP-bound states of Gα13. Furthermore, we find that Itk interacts with Gα13 via the zinc binding regions within its Tec homology domain. The interaction between Itk and Gα13 also results in tyrosine phosphorylation of Gα13, however this is not required for the interaction. Itk enhances Gα13 mediated activation of serum response factor (SRF) transcriptional activity dependent on its ability to interact with Gα13, but its kinase activity is not required to enhance SRF activity. These data reveal a new pathway regulated by Itk in cells, and suggest cross talk between Itk and G-protein signaling downstream of the TcR.