JAK and Src tyrosine kinase signaling in asthma.

Tyrosine kinases play a critical role in transducing intracellular signals from the receptors. Many receptors do not have intrinsic tyrosine kinase activity, so they rely on cytosolic and/or membrane-associated tyrosine kinases for initial signal generation. The Src and JAK family kinases are frequently associated with receptors and generate the initial cytosolic signals. These signals are then transduced to other compartments of the cytosol and to the nucleus to elicit a specific cellular response. In this review we focus on these two families of tyrosine kinases and review their involvement in activation of cells that are involved in the pathogenesis of asthma. A Th2-type immune response dominates the processes that lead to the phenotype of asthma. For this reason we give special attention to the tyrosine kinases that are involved in a Th2 response. Further we examine the involvement of tyrosine kinases in activation of mast cells, eosinophils and other cells.

Establishment of extracellular signal-regulated kinase 1/2 bistability and sustained activation through Sprouty 2 and its relevance for epithelial function.

Our objective was to establish an experimental model of a self-sustained and bistable extracellular signal-regulated kinase 1/2 (ERK1/2) signaling process. A single stimulation of cells with cytokines causes rapid ERK1/2 activation, which returns to baseline in 4 h. Repeated stimulation leads to sustained activation of ERK1/2 but not Jun N-terminal protein kinase (JNK), p38, or STAT6. The ERK1/2 activation lasts for 3 to 7 days and depends upon a positive-feedback mechanism involving Sprouty 2. Overexpression of Sprouty 2 induces, and its genetic deletion abrogates, ERK1/2 bistability. Sprouty 2 directly activates Fyn kinase, which then induces ERK1/2 activation. A genome-wide microarray analysis shows that the bistable phospho-ERK1/2 (pERK1/2) does not induce a high level of gene transcription. This is due to its nuclear exclusion and compartmentalization to Rab5+ endosomes. Cells with sustained endosomal pERK1/2 manifest resistance against growth factor withdrawal-induced cell death. They are primed for heightened cytokine production. Epithelial cells from cases of human asthma and from a mouse model of chronic asthma manifest increased pERK1/2, which is associated with Rab5+ endosomes. The increase in pERK1/2 was associated with a simultaneous increase in Sprouty 2 expression in these tissues. Thus, we have developed a cellular model of sustained ERK1/2 activation, which may provide a mechanistic understanding of self-sustained biological processes in chronic illnesses such as asthma.

MED220/thyroid receptor-associated protein 220 functions as a transcriptional coactivator with Pit-1 and GATA-2 on the thyrotropin-beta promoter in thyrotropes.

Mediator (MED) 220/thyroid receptor-associated protein (TRAP) 220 is a transcriptional mediator that interacts with liganded thyroid/steroid hormone receptors. MED220 haploinsufficient heterozygotes exhibited hypothyroidism and reduced TSHbeta transcripts, suggesting a specific function for TSHbeta transcription. We previously demonstrated that Pit-1 and GATA-2 can bind to a composite element within the proximal TSHbeta promoter and synergistically activate transcription. We detected MED220 expression in TtT-97 thyrotropes by Northern and Western blot analysis. Cotransfections in CV-1 cells showed that Pit-1, GATA-2, or MED220 alone did not markedly stimulate the TSHbeta promoter. However, Pit-1 plus GATA-2 resulted in an 10-fold activation, demonstrating synergistic cooperativity. Titration of MED220 resulted in a further dose-dependent stimulation up to 25-fold that was promoter specific. Glutathione-S-transferase interaction studies showed that MED220 or GATA-2 each bound the homeodomain of Pit-1, whereas MED220 interacted independently with each zinc finger of GATA-2 but not with either terminus. MED220 interacted with GATA-2 and Pit-1 over a broad region of its N terminus. These regions of interaction were also important for maximal function. Coimmunoprecipitation confirmed that all three factors can interact in thyrotropes and chromatin immunoprecipitation demonstrated in vivo occupancy on the proximal TSHbeta promoter. Thus, the TSHbeta gene is maximally activated by a combination of three thyrotrope transcription factors that act via both protein-DNA and protein-protein interactions.