The Janus kinase/signal transducer and activator of transcription (JAK–STAT) pathway mediates important responses in immune cells. Activation of any of the four JAK family members leads to phosphorylation of one or more of seven STAT family members. Phosphorylation of STAT family members leads to their dimerization and translocation into the nucleus, in which they bind specific DNA sequences to activate gene transcription. Regulation of JAKs and STATs therefore has a significant effect on signal transduction and subsequent cellular responses. Mast cells are important mediators of allergic disease and asthma. These cells have the ability to cause profound inflammation and vasodilation upon the release of preformed mediators, as well as subsequent synthesis of new inflammatory mediators. The regulation of mast cells is therefore of intense interest for the treatment of allergic disease. An important regulator of mast cells, STAT5, is activated downstream of the receptors for immunoglobulin E, interleukin-3 and stem cell factor. STAT5 contributes to mast cell homeostasis, by mediating proliferation, survival, and mediator release. Regulators of the JAK–STAT pathway, such as the suppressors of cytokine signaling (SOCS) and protein inhibitor of activated STAT (PIAS) proteins, are required to fine tune the immune response and maintain homeostasis. A better understanding of the role and regulation of JAKs and STATs in mast cells is vital for the development of new therapeutics.

We previously demonstrated that Transforming Growth Factor (TGF) β1 suppresses IgE-mediated signaling in human and mouse mast cells in vitro, an effect that correlated with decreased expression of the high affinity IgE receptor, FcεRI. The in vivo effects of TGFβ1 and the means by which it suppresses mast cells have been less clear. The current study shows that TGFβ1 suppresses FcεRI and c-Kit expression in vivo. By examining changes in cytokine production concurrent with FcεRI expression, we found that TGFβ1 suppresses TNF production independent of FcεRI levels. Rather, IgE-mediated signaling was altered. TGFβ1 significantly reduced expression of Fyn and Stat5, proteins critical for cytokine induction. These changes may partly explain the effects of TGFβ1, since Stat5B overexpression blocked TGF-mediated suppression of IgE-induced cytokine production. We also found that Stat5B is required for mast cell migration toward SCF, and that TGFβ1 reduced this migration. We found evidence that genetic background may alter TGF responses. TGFβ1 greatly reduced mast cell numbers in Th1-prone C57BL/6 but not Th2-prone 129/Sv mice. Furthermore, TGFβ1 did not suppress IgE-induced cytokine release, and increased c-Kit-mediated migration in 129/Sv mast cells. These data correlated with high basal Fyn and Stat5 expression in 129/Sv cells, which was not reduced by TGFβ1 treatment. Finally, primary human mast cell populations also showed variable sensitivity to TGFβ1-mediated changes in Stat5 and IgE-mediated IL-6 secretion. We propose that TGFβ1 regulates mast cell homeostasis, and that this feedback suppression may be dependent upon genetic context, predisposing some individuals to atopic disease.

Mast cell development is an important component of atopic and chronic inflammatory diseases such as asthma, multiple sclerosis, rheumatoid arthritis, and atherosclerosis. In this study, we found that IL-4 and IL-10 were produced constitutively in cultures of developing mast cells, correlating with mast cell purity. Deletion of either gene increased mast cell numbers and Fc epsilon RI expression during culture in IL-3 + stem cell factor (SCF). By adding exogenous IL-4 and IL-10 to bone marrow (BM) cultures containing IL-3 + SCF, we found that IL-4 + IL-10 suppressed mast cell development through mechanisms not used by either cytokine alone. IL-4 + IL-10 elicited a rapid cell death coincidental with reduced Kit receptor expression and signaling and enhanced mitochondrial damage and caspase activation. IL-4 or IL-10 costimulation, unlike either cytokine alone, altered mast cell ontogeny to yield predominantly macrophages in cultures that typically produce mast cells. This effect was observed consistently with unseparated BM cells, purified mouse BM stem cells, and erythrocyte-depleted human umbilical cord blood cells. These experiments demonstrated a major role for Stat6 and Stat3, but not the Stat3-induced transcriptional repressor Ets variant gene 3. Genetic background was also a critical factor, as BALB/c-derived BM cells were completely resistant to IL-10-mediated killing and expressed lower levels of IL-10R. Collectively, these results support the theory that IL-4 and IL-10 function as endogenous regulators of mast cell progenitor development, consistent with a role in immune homeostasis. Loss of this homeostasis, perhaps via genetic polymorphism, could contribute to the etiology of mast cell-associated disease.

Mast cell responses can be altered by cytokines, including those secreted by Th2 and regulatory T cells (Treg). Given the important role of mast cells in Th2-mediated inflammation and recent demonstrations of Treg-mast cell interactions, we examined the ability of IL-4 and TGF-β1 to regulate mast cell homeostasis. Using in vitro and in vivo studies of mouse and human mast cells, we demonstrate that IL-4 suppresses TGF-β1 receptor expression and signaling, and vice versa. In vitro studies demonstrated that IL-4 and TGF-β1 had balancing effects on mast cell survival, migration, and FcεRI expression, with each cytokine cancelling the effects of the other. However, in vivo analysis of peritoneal inflammation during Nippostrongylus brasiliensis infection in mice revealed a dominant suppressive function for TGF-β1. These data support the existence of a cytokine network involving the Th2 cytokine IL-4 and the Treg cytokine TGF-β1 that can regulate mast cell homeostasis. Dysregulation of this balance may impact allergic disease and be amenable to targeted therapy.

Bile acids are required for intestinal absorption and biliary solubilization of cholesterol and lipids. In addition, bile acids play a crucial role in cholesterol homeostasis. One of the key enzymes in the bile acid biosynthetic pathways is cholesterol 7␣-hydroxylase/cytochrome P450 7␣-hydroxylase (7␣-hydroxylase), which is the rate-limiting and regulatory step of the "classic" pathway. Transcription of the 7␣-hydroxylase gene is highly regulated. Two nuclear receptors, hepatocyte nuclear factor 4␣ (HNF-4␣) and ␣ 1 -fetoprotein transcription factor, are required for both transcription and regulation by different physiological events. It has been shown that some mitogen-activated protein kinases, such as the c-Jun N-terminal kinase and the ERK, play important roles in the regulation of 7␣-hydroxylase transcription. In this study, we show evidence that the p38 kinase pathway plays an important role in 7␣-hydroxylase expression and hence in bile acid synthesis. Inhibition of p38 kinase activity in primary hepatocytes results in ϳ5-10-fold reduction of 7␣-hydroxylase mRNA. This suppression is mediated, at least in part, through HNF-4␣. Inhibition of p38 kinase activity diminishes HNF-4␣ nuclear protein levels and its phosphorylation in vivo and in vitro, and it renders a less stable protein. Induction of the p38 kinase pathway by insulin results in an increase in HNF-4␣ protein and a concomitant induction of 7␣-hydroxylase expression that is blocked by inhibiting the p38 pathway. These studies show a functional link between the p38 signaling pathway, HNF-4␣, and bile acid synthesis.