The initiation and persistence of inflammation is tightly regulated by cell surface receptors on immune cells that function to both amplify the immune response and clear the offending agent. The persistence of inflammation may result in disease inclusive of autoimmune, chronic allergies and asthmatic diseases. One common feature of these diseases is the central role of Fc receptors and immune complexes in the persistence of disease. A second feature is the persistence of high levels of cytokines which serve to recruit other immune cells amplifying the response. Our interest is in understanding how Fc receptors communicate with the nucleus to initiate cytokine responses and how the released cytokines contribute to inflammation. The immediate objective is to identify the molecular events that activate cytokine gene transcription and to evaluate their suitability as targets in therapeutic intervention of disease. We chose to work with mast cells because they are key participants in many inflammatory responses, they express the well-characterized high affinity receptor for IgE (Fc epsilon RI), and they respond to immune complex occupation of the Fc epsilon RI by producing and secreting a diverse array of cytokines and other allergic mediators. Our prior work identified two major families of proteins that link the Fc epsilon RI to nuclear events. The first is the protein kinase C family which is comprised of twelve serine/threonine kinases of which five are known to be expressed in mast cells. Our studies demonstrated that of the five distinct isoforms found in mast cells, two isoforms, beta and epsilon, specifically regulate the synthesis of the AP-1 constituents c-fos and c-jun. One isoform, PKC delta, phosphorylates the Fc epsilon RI and may serve to regulate the receptors ability to communicate intracellularly. These results demonstrate that the different PKC isoforms have specific functions in mast cell responses and thus, makes it more likely that therapeutic targeting of a specific isoform(s) that regulates cytokine gene expression may be possible. The second family of proteins we are studying is that of guanine nucleotide exchange factors (GEF). These proteins facilitate the exchange of guanine diphosphate (GDP) to guanine trisphosphate (GTP) from small GTP-binding proteins which serves to activate the latters function in regulating numerous signaling pathways. We found that a member of the GEF family called Vav, which is expressed only in hematopoietic cells, may link the Fc epsilon RI to nuclear events, since it is activated in these cells upon Fc epsilon RI stimulation and serves to activate mitogen-activated protein kinase (MAPK) family members that are known to stimulate gene transcription. Prior evidence from our lab and and that of others demonstrated that Vav contributes to the activation of cytokine gene transcription, in particular to the induction of interleukin-2 (IL-2). As a hematopoietic cell-specific protein, Vav may serve a unique role in immune cells, and investigating its functional role in these cells may identify new areas with therapeutic potential.In the past year our objectives were: 1. To determine the significance of the Fc epsilon RI phosphorylation mediated by PKC delta to mast cell responses. 2. To study the role of Vav in the induction of cytokine gene expression. 3. To determine where Vav is localized in the cell and the importance of this localization to Vav function. We met these objectives in the following manner: We identified the site of phosphorylation of the Fc epsilon RI gamma chain by PKC delta and found that mutation of this threonine 60 (T60) to alanine (A) resulted in a defective Fc epsilon RI mediated TNF alpha production and in the inhibition of antigen clearance from the mast cell surface. In the analysis of affected signaling pathways we found that mast cells with the T60A mutated gamma chain were defective in activation of Syk kinase, a kinase essential to mast cell degranulation and cytokine production. By substituting an aspartic acid (D) residue at T60 we demonstrated that a negative charge is required at T60 to efficiently activate Syk. These findings demonstrate that the activation of Syk kinase is dependent on the negative charge provided by threonine phosphorylation of the Fc epsilon RI gamma chain in these cells and identifies a new area with therapeutic potential in allergic diseases. Other studies showed that Vav stimulates an increase in IL-2 mRNA in mast cells, an observation previously reported for the role of Vav in T-cells. However, novel to our studies is the role of Vav in stimulating large increases in mRNA for IL-6. This increase in IL-6 mRNA is dependent on the activation of c-jun N terminal kinase (JNK) by Vav via the activation of the small GTP-binding protein Rac 1 by Vav. We also found that the small GTP-binding protein Ras does not contribute to the Vav-mediated induction of IL-6. In addition, overexpression of Vav in primary mast cells results in increased secretion of IL-6 from these cells. Thus, these findings show that Vav/JNK activity is important to the production of IL-6. In addition, we used confocal microscopy to localize the cellular site of Vav activity to the plasma membrane. In response to Fc epsilon RI-stimulation we found that Vav redistributes from the cytoplasm to a submembraneous location forming small micro-aggregates which co-localize with the Fc epsilon RI. The localization of Vav to these sites is dependent on the SH2 domain of Vav (a protein domain that recognizes proteins phosphorylated on tyrosine residues) and this localization is important to Vavs ability to activate JNK. We also discovered that Vav localizes to specialized regions on the plasma membrane that are enriched in gycosphingolipids and cholesterol. These glycosphingolipid-enriched microdomains (GEMs) are thought to be possible sites where receptors may engage the cellular signaling apparatus responsible for initiating a cells response to a stimulus. Our studies demonstrated that both the Fc epsilon RI and Vav are found in these domains and that the presence of Vav in these domains is critical for its function. Thus, we provide strong evidence for the GEMs as sites where receptors engage downstream effectors. This may provide new areas with therpeutic potential. In summary, we found that the threonine phosphorylation of the Fc epsilon RI gamma chain is important to the activation of Syk kinase, an enzyme essential to mast cell degranulation and cytokine production. We identified the site of Vav function in mast cells and found that it specifically regulates IL-6, a cytokine important in inflammatory disease. We also found that Vav localization to specialized membrane domains (GEMs) is critical to its function. Thus these findings may provide novel targets for therapeutic intervention in inflammatory diseases.
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