Little is known about the signals downstream of PI3K which regulate mast cell homeostasis and function following FcεRI aggregation and Kit ligation. In this study, we investigated the role of the mammalian target of rapamycin complex 1 (mTORC1) pathway in these responses. In human and mouse mast cells, stimulation via FcεRI or Kit resulted in a marked PI3K-dependent activation of the mTORC1 pathway, as revealed by the wortmannin-sensitive sequential phosphorylation of tuberin, mTOR, p70S6 kinase (p70S6K), and 4E-BP1. In contrast, in human tumor mast cells, the mTORC1 pathway was constitutively activated and this was associated with markedly elevated levels of mTORC1 pathway components. Rapamycin, a specific inhibitor of mTORC1, selectively and completely blocked the FcεRI- and Kit-induced mTORC1-dependent p70S6K phosphorylation and partially blocked the 4E-BP1 phosphorylation. In parallel, although rapamycin had no effect on FcεRI-mediated degranulation or Kit-mediated cell adhesion, it inhibited cytokine production, and kit-mediated chemotaxis and cell survival. Furthermore, Rapamycin also blocked the constitutive activation of the mTORC1 pathway and inhibited cell survival of tumor mast cells. These data provide evidence that mTORC1 is a point of divergency for the PI3K-regulated downstream events of FcεRI and Kit for the selective regulation of mast cell functions. Specifically, the mTORC1 pathway may play a critical role in normal and dysregulated control of mast cell homeostasis.

Mast cells play a central role in the initiation of inflammatory responses associated with asthma and other allergic disorders. Receptor-mediated mast cell growth, differentiation, homing to their target tissues, survival, and activation, are all controlled, to varying degrees, by phosphoinositide-3-kinase (PI3K)-driven pathways. It is not fully understood how such diverse responses can be differentially regulated by PI3K. However, recent studies have provided greater insight into the mechanisms which control, and those which are controlled by, different PI3K subunit isoforms in mast cells. In this review, we discuss how PI3K influences the mast cell processes described above. Furthermore, we describe how different mast cell receptors utilize alternative isoforms of PI3K for these functions, and discuss potential downstream targets of these isoforms.

Antigen/IgE-mediated mast cell activation via FcεRI can be markedly enhanced by the activation of other receptors expressed on mast cells and these receptors may thus contribute to the allergic response in vivo. One such receptor family is the G protein-coupled receptors (GPCRs). Although the signaling cascade linking FcεRI aggregation to mast cell activation has been extensively investigated, the mechanisms by which GPCRs amplify this response are relatively unknown. To investigate this, we utilized prostaglandin (PG)E 2 based on initial studies demonstrating its greater ability to augment antigen-mediated degranulation in mouse mast cells than other GPCR agonists examined. This enhancement, and the ability of PGE 2 to amplify antigen-induced calcium mobilization, was independent of phosphoinositide 3-kinase but was linked to a pertussis toxinsensitive synergistic translocation to the membrane of phospholipase (PL)Cγ and PLCβ and to an enhancement of PLCγ phosphorylation. This "trans-synergistic" activation of PLCβ and γ, in turn, enhanced production of inositol 1,4,5-trisphosphate, store operated calcium entry, and activation of protein kinase C (PKC) (α and β). These responses were critical for the promotion of degranulation. This is the first report of synergistic activation between PLCγ and PLCβ that permits reinforcement of signals for degranulation in mast cells.

Activated mast cells are a major source of the eicosanoids PGD2 and leukotriene C4 (LTC4), which contribute to allergic responses. These eicosanoids are produced following the ERK1/2-dependent activation of cytosolic phospholipase A2, thus liberating arachidonic acid, which is subsequently metabolized by the actions of 5-lipoxygenase and cyclooxygenase to form LTC4 and PGD2, respectively. These pathways also generate reactive oxygen species (ROS), which have been proposed to contribute to FcεRI-mediated signaling in mast cells. In this study, we demonstrate that, in addition to ERK1/2-dependent pathways, ERK1/2-independent pathways also regulate FcεRI-mediated eicosanoid and ROS production in mast cells. A role for the Tec kinase Btk in the ERK1/2-independent regulatory pathway was revealed by the significantly attenuated FcεRI-dependent PGD2, LTC4, and ROS production in bone marrow-derived mast cells of Btk−/− mice. The FcεRI-dependent activation of Btk and eicosanoid and ROS generation in bone marrow-derived mast cells and human mast cells were similarly blocked by the PI3K inhibitors, Wortmannin and LY294002, indicating that Btk-regulated eicosanoid and ROS production occurs downstream of PI3K. In contrast to ERK1/2, the PI3K/Btk pathway does not regulate cytosolic phospholipase A2 phosphorylation but rather appears to regulate the generation of ROS, LTC4, and PGD2 by contributing to the necessary Ca2+ signal for the production of these molecules. These data demonstrate that strategies to decrease mast cell production of ROS and eicosanoids would have to target both ERK1/2- and PI3K/Btk-dependent pathways.

Increased mast cell burden is observed in the inflamed tissues and affected organs and tissues of patients with mast cell proliferative disorders. However, normal mast cells participate in host defense, so approaches to preferentially target clonally expanding mast cells are needed. We found that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) are up-regulated in neoplastic and developing immature mast cells compared with their terminally differentiated counterparts. Elevated mTOR mRNA was also observed in bone marrow mononuclear cells of patients exhibiting mast-cell hyperplasia. Selective inhibition of mTORC1 and mTORC2 through genetic and pharmacologic manipulation revealed that, whereas mTORC1 may contribute to mast-cell survival, mTORC2 was only critical for homeostasis of neoplastic and dividing immature mast cells. The cytostatic effect of mTORC2 down-regulation in proliferating mast cells was determined to be via inhibition of cell-cycle progression. Because mTORC2 was observed to play little role in the homeostasis of differentiated, nonproliferating, mature mast cells, these data provide a rationale for adopting a targeted approaching selectively inhibiting mTORC2 to effectively reduce the proliferation of mast cells associated with inflammation and disorders of mast cell proliferation while leaving normal differentiated mast cells largely unaffected. IntroductionMast cells (MCs) are considered to be critical components of both the innate and acquired immune defense systems. 1 Central to these functions is the ability of MCs to release a plethora of inflammatory mediators after activation through cell-surface receptors, primarily the high-affinity receptors for IgE (Fc⑀RI). 2 Although these reactions are considered to have evolved to protect host organisms against invading parasites and other microorganisms, 3 inappropriate or exaggerated activation of MCs manifests the reactions associated with allergic diseases.MCs develop from bone marrow (BM) CD13 ϩ /CD34 ϩ /CD117 (KIT) ϩ progenitor cells that enter into circulation and mature during migration to and residency in their target tissues. 4 MC numbers within tissues appear to be tightly regulated, with a several-fold increase in numbers occurring in inflammatory conditions and even higher numbers in association with parasitic inflammation. Clonal MC disorders may result in 10-fold or greater numbers of MCs in tissues such as the BM, liver, and spleen. [5][6] Similarly, a dysregulated increase in MC numbers is also observed in certain types of cancer, and in that context may contribute to cancer progression. [7][8][9] We observed previously that the mammalian target of rapamycin (mTOR) is overexpressed and constitutively phosphorylated in neoplastic MCs regardless of whether activating mutations in the MC growth factor receptor KIT are present. 10 MTOR is a serine/ threonine kinase that regulates divergent signaling pathways depending on its interactions with 2 regulatory proteins: raptor, a major component of mTOR complex 1 (mTORC1), a...

Various growth factors and cytokines on the regulation of type I collagen and hyaluronan in human dermal skin probably function as key factors in skin remodeling and skin aging. Our profile may help to apply to cosmeceutical area maintaining as young skin through the increase in extracellular matrix.

In addition to regulating mast cell homeostasis, the activation of KIT following ligation by stem cell factor promotes a diversity of mast cell responses, including cytokine production and chemotaxis. Although we have previously defined a role for the mammalian target of rapamycin complex 1 in these responses, it is clear that other signals are also required for maximal KIT-dependent cytokine production and chemotaxis. In this study, we provide evidence to support a role for glycogen synthase kinase 3β (GSK3β) in such regulation in human mast cells (HuMCs). GSK3β was observed to be constitutively activated in HuMCs. This activity was inhibited by knockdown of GSK3β protein following transduction of these cells with GSK3β-targeted shRNA. This resulted in a marked attenuation in the ability of KIT to promote chemotaxis and, in synergy with FcεRI-mediated signaling, cytokine production. GSK3β regulated KIT-dependent mast cell responses independently of mammalian target of rapamycin. However, evidence from the knockdown studies suggested that GSK3β was required for activation of the MAPKs, p38, and JNK and downstream phosphorylation of the transcription factors, Jun and activating transcription factor 2, in addition to activation of the transcription factor NF-κB. These studies provide evidence for a novel prerequisite priming mechanism for KIT-dependent responses regulated by GSK3β in HuMCs.

During aging, the structural changes of epidermis and dermis have been well established including matrix expression, alteration, and thickness by analyzing skin biopsies. 1,2 However, little is known about the anatomical changes of subcutaneous fat layer with age. That is probably why the layer is located deeper to obtain the sample biopsy in the skin. To our knowledge, the skin adipose tissue participates in energy storage, thermogenesis, endocrine secretion, and immune defense as well. In recent reports, it was shown that the facial adipose tissue might be a potential target against the signs of facial aging such as elasticity loss and wrinkle formation. 3,4 Although subcutaneous fat loss has been considered to be the typical hallmark of facial aging, 5 it was demonstrated that the infraorbital fat of old group was significantly thicker than that of young group in Asian woman using computed tomography. 6 A high-resolution diagnostic ultrasound system has been used to evaluate deep dermal and subcutaneous thickness. 7,8 It was suggested that ultrasound system provides the most accurate and reliable technique for the measurement of subcutaneous fat layer.