Stem cell factor-induced migration of mast cells requires p38 mitogen-activated protein kinase activity.

Stem cell factor (SCF) can be considered a cardinal cytokine in mast cell biology as it affects mast cell differentiation, survival, and migration. The objective of this study was to investigate the role of two mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) and p38, in SCF-induced cell migration. This was examined in mouse mast cells by using PD 098059 and SB203580, which are specific inhibitors of mitogen-induced extracellular kinase (MEK) and p38 MAP kinase, respectively. SCF induced a rapid and transient activation of ERK and p38 in a dose-dependent manner. Inhibition of p38 activity by SB203580 was paralleled with a marked reduction of migration toward SCF, whereas the effect of the MEK inhibitor was less pronounced. This is the first report of a physiological function of SCF-dependent activation of p38. Whether p38-mediated mast cell migration is a possible target for suppression of mast cell hyperplasia remains to be determined.     

Sensitized mast cells migrate toward the antigen: a response regulated by p38 mitogen-activated protein kinase and Rho-associated coiled-coil-forming protein kinase

Although mast cells accumulate within the mucosal epithelial layer of patients with allergic rhinitis and bronchial asthma, the responsible chemotactic factors are undefined. We investigated whether mast cells sensitized with Ag-specific IgE migrate toward the Ag. MC/9 mast cells sensitized with anti-DNP IgE migrated toward DNP-conjugated human serum albumin. This migration was directional, and the degree was stronger than that induced by stem cell factor. IL-3 and stem cell factor-dependent cultured mast cells derived from mouse bone marrow also migrated toward the Ag. Subsequent migration mediated by the Fc(epsilon)RI was significantly inhibited by incubating the cells with Y-27632, a Rho-associated coiled-coil-forming protein kinase inhibitor, or with SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Both p38 MAPK and MAPK-activated protein kinase (MAPKAPK)2 were activated following Fc(epsilon)RI aggregation, and activation of MAPKAPK2 was almost completely inhibited by 10 microM SB203580. Wortmannin or a low concentration of SB203580 partially inhibited MAPKAPK2, but did not block mast cell migration. In contrast, Y-27632 did not affect the activation of MAPKAPK2. These results indicate that Ag works not only as a stimulant for allergic mediators from IgE-sensitized mast cells, but also as a chemotactic factor for mast cells. Both p38 MAPK activation and Rho-dependent activation of Rho-associated coiled-coil-forming protein kinase may be required for Fc(epsilon)RI-mediated cell migration.     

Tyrosine kinase-deficient Wv c-kit induces mast cell adhesion and chemotaxis

W/W^v mice are deficient intissue mast cells, and mast cells cultured from these mice do notproliferate in response to the c-kit ligand, stem cell factor (SCF). Inthis paper, we report that mouse bone marrow cultured mast cellsderived from W/W^v mice do adhereto fibronectin in the presence of SCF and exhibit chemotaxis to SCF,and we explore this model for the understanding of c-kit-mediatedsignaling pathways. Both in vitro and in vivo (in intact cells)phosphorylation experiments demonstrated a low residual level ofW/W^v c-kit proteinphosphorylation. SCF-induced responses inW/W^v mast cells were abolished bythe tyrosine kinase inhibitor herbimycin A and by thephospatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin butwere not affected by protein kinase C inhibitors. These observationsare consistent with the conclusions thatW^v c-kit initiates a signalingprocess that is PI 3-kinase dependent and that mutatedW^v c-kit retains the ability toinitiate mast cell adhesion and migration.

     

Fps/Fes protein-tyrosine kinase regulates mast cell adhesion and migration downstream of Kit and β1 integrin receptors

Activation of Kit receptor protein-tyrosine kinase (PTK) by its ligand Stem Cell Factor (SCF) is required for the development of mast cells, and for the regulation of mast cell proliferation, migration and modulation of inflammatory mediator release. Recent studies have implicated the non-receptor PTK Fps/Fes (hereafter referred to as Fes) in signaling downstream of oncogenic Kit, however, the potential role of Fes in regulating Kit signaling is not well defined. In this study, we show that SCF induces transient tyrosine phosphorylation of wild-type Fes as well as kinase-dead Fes in bone marrow-derived mast cells (BMMCs). The latter finding implicates an upstream kinase acting on Fes, which we identified as Fyn PTK. SCF treatment of BMMCs promoted recruitment of Fes to Kit, potentially via direct interaction of the Fes SH2 domain with phosphorylated Kit. While Fes was not required for SCF-induced signaling to Akt and Erk kinases, Fes-deficient (fes^?/?) BMMCs displayed a defect in sustained p38 kinase activation, compared to control cells. SCF-treated Fes-deficient BMMCs also displayed elevated β1 integrin-mediated cell adhesion and spreading on fibronectin, compared to control cells, and a reduction in cell polarization at later times of SCF treatment. Restoring Fes expression in fes^?/? BMMCs by retroviral transduction was sufficient to rescue cell spreading and polarization defects. Interestingly, SCF-induced chemotaxis of BMMCs was also defective in Fes-deficient BMMCs, and restored in Fes-rescue BMMCs. Overall, these results implicate Fes in regulating cross-talk between Kit and β1 integrins to promote cytoskeletal reorganization and motility of mast cells.     

Increased Kit/SCF receptor induced mitogenicity but abolished cell motility after inhibition of protein kinase C.

The product of the c-kit proto-oncogene, denoted Kit/SCF-R, encodes a tyrosine kinase receptor for stem cell factor (SCF). Kit/SCF-R induces proliferation, differentiation or migration of cells within the hematopoietic, gametogenic and melanogenic lineages at different developmental stages. We report here that protein kinase C (PKC) mediates phosphorylation of Kit/SCF-R on serine residues in response to SCF or PMA in intact cells. The phosphorylation inhibits SCF-induced tyrosine autophosphorylation of Kit/SCF-R. In vitro studies showed that PKC phosphorylated the Kit/SCF-R directly on serine residues and inhibited autophosphorylation of Kit/SCF-R, as well as its kinase activity towards an exogenous substrate. The PKC-induced phosphorylation did not affect Kit/SCF-R ligand binding affinity. Inhibition of PKC led to increased SCF-induced tyrosine autophosphorylation, as well as increased SCF-induced mitogenicity. In contrast, PKC was necessary for SCF-induced motility responses, including actin reorganization and chemotaxis. Our data suggest that PKC is involved in a negative feedback loop which regulates the Kit/SCF-R and that the activity of PKC determines whether the effect of SCF will be preferentially mitogenic or motogenic.     

C-Kit Promotes Growth and Migration of Human Cardiac Progenitor Cells via the PI3K-AKT and MEK-ERK Pathways

A recent phase I clinical trial (SCIPIO) has shown that autologous c-kit+ cardiac progenitor cells (CPCs) improve cardiac function and quality of life when transplanted into patients with ischemic heart disease. Although c-kit is widely used as a marker of resident CPCs, its role in the regulation of the cellular characteristics of CPCs remains unknown. We hypothesized that c-kit plays a role in the survival, growth, and migration of CPCs. To test this hypothesis, human CPCs were grown under stress conditions in the presence or absence of SCF, and the effects of SCF-mediated activation of c-kit on CPC survival/growth and migration were measured. SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions. In addition, SCF significantly promoted CPC migration in vitro. Furthermore, the pro-survival and pro-migratory effects of SCF were augmented by c-kit overexpression and abrogated by c-kit inhibition with imatinib. Mechanistically, c-kit activation in CPCs led to activation of the PI3K and the MAPK pathways. With the use of specific inhibitors, we confirmed that the SCF/c-kit-dependent survival and chemotaxis of CPCs are dependent on both pathways. Taken together, our findings suggest that c-kit promotes the survival/growth and migration of human CPCs cultured ex vivo via the activation of PI3K and MAPK pathways. These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit.     

Stem cell factor has a suppressive activity to IgE-mediated chemotaxis of mast cells

Stem cell factor (SCF), which is well known as a cytokine capable of amplifying development and functions of mast cells, is mainly released from fibroblasts in the peripheral tissue. To investigate whether SCF controlled chemotactic migration of mast cells induced by IgE-specific Ag, murine bone marrow-derived cultured mast cells (BMCMC) and human cord blood-derived cultured mast cells (HuCMC) were preincubated with SCF. Although BMCMC and HuCMC sensitized with IgE directly moved toward specific Ag, preincubation for even 1 h with an optimal dose of SCF suppressed the IgE-mediated chemotactic movement. No or little inhibitory effect of SCF was detected in BMCMC derived from c-kit receptor-defect WBB6F1-W/Wv mice. In contrast, preincubation of BMCMC and HuCMC with SCF enhanced beta-hexosaminidase release and Ca2+ mobilization in response to Ag after sensitization with IgE. Using the real-time record of chemotactic migration, BMCMC preincubated with SCF manifested motionless without degranulation. These results suggest that locally produced SCF may have an inhibitory effect on chemotaxis of mast cells, contributing to their accumulation and enhancement of functions at the peripheral site in allergic and nonallergic conditions.     

SCF-induced airway hyperreactivity is dependent on leukotriene production

Stem cell factor (SCF) is directly involved in the induction of airway hyperreactivity during allergen-induced pulmonary responses in mouse models. In these studies, we examined the specific mediators and mechanisms by which SCF can directly induce airway hyperreactivity via mast cell activation. Initial in vitro studies with bone marrow-derived mast cells indicated that SCF was able to induce the production of bronchospastic leukotrienes, LTC(4) and LTE(4). Subsequently, when SCF was instilled in the airways of naive mice, we were able to observe a similar induction of LTC(4) and LTE(4) in the bronchoalveolar lavage (BAL) fluid and lungs of treated mice. These in vivo studies clearly suggested that the previously observed SCF-induced airway hyperreactivity may be related to the leukotriene production after SCF stimulation. To further investigate whether the released leukotrienes were the mediators of the SCF-induced airway hyperreactivity, an inhibitor of 5-lipoxygenase (5-LO) binding to the 5-LO activating protein (FLAP) was utilized. The FLAP inhibitor MK-886, given to the animals before intratracheal SCF administration, significantly inhibited the release of LTC(4) and LTE(4) into the BAL fluid. More importantly, use of the FLAP inhibitor nearly abrogated the SCF-induced airway hyperreactivity. In addition, blocking the LTD(4)/E(4), but not LTB(4), receptor attenuated the SCF-induced airway hyperreactivity. In addition, the FLAP inhibitor reduced other mast-derived mediators, including histamine and tumor necrosis factor. Altogether, these studies indicate that SCF-induced airway hyperreactivity is dependent upon leukotriene-mediated pathways.     

Suppressor of cytokine signaling 6 associates with KIT and regulates KIT receptor signaling

Suppressor of cytokine signaling (SOCS) proteins are a family of Src homology 2-containing adaptor proteins. Cytokine-inducible Src homology domain 2-containing protein, SOCS1, SOCS2, and SOCS3 have been implicated in the down-regulation of cytokine signaling. The function of SOCS4, 5, 6, and 7 are not known. KIT receptor signaling is regulated by protein tyrosine phosphatases and adaptor proteins. We previously reported that SOCS1 inhibited cell proliferation in response to stem cell factor (SCF). By screening the other members of SOCS family, we identified SOCS6 as a KIT-binding protein. Using KIT mutants and peptides, we demonstrated that SOCS6 bound directly to KIT tyrosine 567 in the juxtamembrane domain. To investigate the function of this interaction, we constitutively expressed SOCS6 in cell lines. Ectopic expression of SOCS6 in Ba/F3-KIT cell line decreased cell proliferation in response to SCF but not SCF-induced chemotaxis. SOCS6 reduced SCF-induced activation of ERK1/2 and p38 but not activation of AKT or STATs in Ba/F3, murine embryonic fibroblast (MEF), or COS-7 cells. SOCS6 did not impair ERK and p38 activation by other stimuli. These results indicate that SOCS6 binds to KIT juxtamembrane region, which affects upstream signaling components leading to MAPK activation. Our results indicate that KIT signaling is regulated by several SOCS proteins and suggest a putative function for SOCS6 as a negative regulator of receptor tyrosine kinases.     

RIN3 Is a Negative Regulator of Mast Cell Responses to SCF

Stimulation of the receptor tyrosine kinase KIT by Stem Cell Factor (SCF) triggers activation of RAS and its downstream effectors. Proper KIT activation is essential for the maturation, survival and proliferation of mast cells. In addition, SCF activation of KIT is critical for recruiting mast cells to sites of infection or injury, where they release a mix of pro-inflammatory substances. RIN3, a RAS effector and RAB5-directed guanine nucleotide exchange factor (GEF), is highly expressed and enriched in human mast cells. SCF treatment of mast cells increased the amount of GTP-bound RAB5, and the degree of RAB5 activation correlated with the expression level of RIN3. At the same time, SCF caused the dissociation of a pre-formed complex of RIN3 with BIN2, a membrane bending protein implicated in endocytosis. Silencing of RIN3 increased the rate of SCF-induced KIT internalization, while persistent RIN3 over-expression led to KIT down regulation. These observations strongly support a role for RIN3 in coordinating the early steps of KIT endocytosis. Importantly, RIN3 also functioned as an inhibitor of mast cell migration toward SCF. Finally, we demonstrate that elevated RIN3 levels sensitize mastocytosis cells to treatment with a KIT tyrosine kinase inhibitor, suggesting the value of a two-pronged inhibitor approach for this difficult to treat malignancy. These findings directly connect KIT activation with a mast cell-specific RAS effector that regulates the cellular response to SCF and provide new insight for the development of more effective mastocytosis treatments.     

Spred-1 negatively regulates interleukin-3-mediated ERK/mitogen-activated protein (MAP) kinase activation in hematopoietic cells

Sprouty/Spred family proteins have been identified as negative regulators of growth factor-induced ERK/mitogen-activated protein (MAP) kinase activation. However, it has not been clarified whether these proteins regulate cytokine-induced ERK activity. We found that Spred-1 is highly expressed in interleukin-3 (IL-3)-dependent hematopoietic cell lines and bone marrow-derived mast cells. To investigate the roles of Spred-1 in hematopoiesis, we expressed wild-type Spred-1 and a dominant negative form of Spred-1, DeltaC-Spred, in IL-3- and stem cell factor (SCF)-dependent cell lines as well as hematopoietic progenitor cells from mouse bone marrow by retrovirus gene transfer. In IL-3-dependent Ba/F3 cells expressing c-kit, forced expression of Spred-1 resulted in a reduced proliferation rate and ERK activation in response to not only SCF but also IL-3. In contrast, DeltaC-Spred augmented IL-3-induced cell proliferation and ERK activation. Wild-type Spred-1 inhibited colony formation of bone marrow cells in the presence of cytokines, whereas DeltaC-Spred-1 expression enhanced colony formation. Augmentation of ERK activation and proliferation in response to IL-3 was also observed in Spred-1-deficient bone marrow-derived mast cells. These data suggest that Spred-1 negatively regulates hematopoiesis by suppressing not only SCF-induced but also IL-3-induced ERK activation.     

Stem cell factor (SCF) can regulate the activation and expansion of murine intraepithelial lymphocytes

Murine intraepithelial lymphocytes (IEL) express c-kit, the receptor for stem cell factor (SCF). SCF induced a low but significant proliferative response in IEL, but not in splenic T cells. SCF stimulation of IEL resulted in an expansion of the c-kit(+), TCRgammadelta(+)cell population. SCF-induced proliferation was dependent upon SCF-c-kit interactions, since antibody to c-kit blocked this response, and IEL obtained from c-kit mutant (W/W(v)) mice failed to respond to SCF. SCF acted synergistically with anti-TCRgammadelta and with concavalin A (Con A) to induce proliferation and interferon gamma (IFN-gamma) production in IEL. Finally, mice injected with SCF had a significant increase in the number of IEL in the small intestine. SCF-treated mice had increased numbers of TCRalphabeta(+)and TCRgammadelta(+)cell populations, as well as increased numbers of c-kit(+)and c-kit(-)IEL. These data suggest that SCF-c-kit interactions play an important role in regulating IEL expansion and activation.     

Protein-tyrosine phosphatase alpha regulates stem cell factor-dependent c-Kit activation and migration of mast cells

The role of protein-tyrosine phosphatase alpha (PTPalpha) in mast cell function was investigated in tissues and cells from PTPalpha-deficient mice. Bone marrow-derived mast cells (BMMCs) lacking PTPalpha exhibit defective stem cell factor (SCF)-dependent polarization and migration. Investigation of the molecular basis for this reveals that SCF/c-Kit-stimulated activation of the Fyn tyrosine kinase is impaired in PTPalpha(-/-) BMMCs, with a consequent inhibition of site-specific c-Kit phosphorylation at tyrosines 567/569 and 719. Although c-Kit-mediated activation of phosphatidylinositol 3-kinase and Akt is unaffected, profound defects occur in the activation of downstream signaling proteins, including mitogen-activated protein kinases and Rho GTPases. Phosphorylation and interaction of Fyn effectors Gab2 and Shp2, which are linked to Rac/JNK activation in mast cells, are impaired in PTPalpha(-/-) BMMCs. Thus, PTPalpha is required for SCF-induced c-Kit and Fyn activation, and in this way regulates a Fyn-based c-Kit signaling axis (Fyn/Gab2/Shp2/Vav/PAK/Rac/JNK) that mediates mast cell migration. These defective signaling events may underlie the altered tissue-resident mast cell populations found in PTPalpha(-/-) mice.     

Stem cell factor induces ERM proteins phosphorylation through PI3K activation to mediate melanocyte proliferation and migration

Stem cell factor (SCF) activates a variety of signals associated with stimulation of proliferation, differentiation, migration, and survival in melanocytes. However, the molecular mechanisms by which SCF and its receptor Kit activates these signaling pathways simultaneously and independently are still poorly defined. Here, we examined whether SCF induces ezrin/radixin/moesin (ERM) proteins phosphorylation as a downstream target of PI3K in melanocytes. ERM proteins are cross-linkers between the plasma membrane and the actin cytoskeleton and are activated by phosphorylation of a C-terminal threonine residue. Our results demonstrated that SCF-induced ERM proteins phosphorylation on threonine residue and Rac1 activation in cultured normal human melanocytes through the activation of PI3K. The functional role of phosphorylated-ERM proteins was examined using melanocytes infected with adenovirus carrying a dominant negative mutant (Ala-558, TA) or wild type of moesin. In the TA moesin-overexpressing melanocytes, SCF-induced cell proliferation and migration were inhibited. Thus, our results indicate that phosphorylation of ERM proteins plays an important role in the regulation of SCF-induced melanocyte proliferation and migration.     

Ailanthus altissima swingle has anti-anaphylactic effect and inhibits inflammatory cytokine expression via suppression of nuclear factor-kappaB activation

Ailanthus altissima swingle (ailanthic cortex, AAS) has been used as a traditional medicine for fever, bleeding, infection, and inflammation for many years in Korea. However, its mechanisms have not been examined. In the present study, we investigate the effect of AAS on the mast-cell-mediated allergic and inflammatory reaction using in vivo and in vitro models and elucidate its molecular mechanisms. AAS significantly inhibited compound 48/48-induced edema and systemic anaphylaxis. AAS significantly inhibited passive cutaneous anaphylaxis. AAS inhibited histamine release from rat peritoneal mast cells (RPMCs) in a dose-dependent manner. Moreover, AAS significantly inhibited production of inflammatory cytokines, tumor necrosis factor (TNF), interleukin (IL)-6, and IL-8 on the phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-stimulated human mast cell line, HMC-1 cells. AAS inhibits the IgE or stem cell factor-induced TNF production on RPMCs. In activated HMC-1 cells, the expression level of NF-kappaB/Rel A protein increased in the nucleus, whereas the level of NF-kappaB/Rel A in the nucleus was decreased by AAS treatment. In addition, AAS inhibited the PMACI-induced IkappaBalpha degradation. In conclusion, the present results indicate that AAS has potent anti-anaphylactic and anti-inflammatory properties.     

Suppression of normal and malignant kit signaling by a bispecific antibody linking kit with CD300a

Through its receptor Kit (CD117), stem cell factor (SCF) critically regulates human mast cell (MC) differentiation, survival, priming, and activation. The dominance of SCF in setting these parameters compels stringent contra-regulation to maintain a balanced MC phenotype. We have synthesized a library of bispecific Ab fragments to examine the effect of linking Kit with CD300a. In this study, we report that CD300a exerts a strong inhibitory effect on Kit-mediated SCF-induced signaling, consequently impairing MC differentiation, survival, and activation in vitro. This effect derives from Kit-mediated tyrosine phosphorylation of CD300a and recruitment of the SHIP-1 but not of SH2-containing protein phosphatase 1. CD300a inhibits the constitutive activation of the human leukemic HMC-1 cells but not their survival. Finally, CD300a abrogates the allergic reaction induced by SCF in a murine model of cutaneous anaphylaxis. Our findings highlight CD300a as a novel regulator of Kit in human MC and suggest roles for this receptor as a suppressor of Kit signaling in MC-related disorders.     

Stem cell factor plays a major role in the recruitment of eosinophils in allergic pleurisy in mice via the production of leukotriene B4

The understanding of the mechanisms underlying eosinophil migration into tissue is an essential step in the development of novel therapies aimed at treating allergic diseases where eosinophil recruitment and activation are thought to play an essential role. In this study, we have examined the effects of the in vivo administration of stem cell factor (SCF) on eosinophil recruitment and tested whether endogenous SCF was involved in mediating eosinophil recruitment in response to Ag challenge in sensitized mice. The intrapleural injection of SCF induced a time- and concentration-dependent recruitment of eosinophils in mice. In allergic mice, SCF message was expressed early after Ag challenge and returned to baseline levels after 8 h. In agreement with the ability of SCF to induce eosinophil recruitment and its expression in the allergic reaction, an anti-SCF polyclonal Ab abrogated eosinophil recruitment when given before Ag challenge. SCF increased the levels of leukotriene B4 (LTB4) in the pleural cavity of mice and an LTB4 receptor antagonist, CP105,696, abrogated the effects of SCF on eosinophil recruitment. Similarly, recruitment of eosinophils in the allergic reaction was virtually abolished by CP105,696. Together, our data favor the hypothesis that the local release of SCF following Ag challenge may activate and/or prime mast cells for IgE-mediated release of inflammatory mediators, especially LTB4. The mediators released in turn drive the recruitment of eosinophils. Inhibition of the function of SCF in vivo may reduce the migration of eosinophils to sites of allergic inflammation and may, thus, be a relevant principle in the treatment of allergic diseases.     

Promotion of cancer cell migration: an insulin-like growth factor (IGF)-independent action of IGF-binding protein-6

A family of six high affinity IGF-binding proteins (IGFBPs 1-6) plays an important role in modulating IGF activities. Recent studies suggest that some IGFBPs may have IGF-independent effects, including induction of apoptosis and modulation of cell migration. However, very little is known about possible IGF-independent actions of IGFBP-6. We have generated a non-IGF-binding IGFBP-6 mutant by substituting Ala for four amino acid residues (Pro(93)/Leu(94)/Leu(97)/Leu(98)) in its N-domain IGF-binding site. A >10,000-fold loss of binding affinity for IGF-I and IGF-II was observed using charcoal solution binding assay, BIAcore biosensor, and ligand blotting. Wild-type and mutant IGFBP-6, as well as IGF-II, induced cell migration in RD rhabdomyosarcoma and LIM 1215 colon cancer cells. Cell migration was mediated by the C-domain of IGFBP-6. Transient p38 phosphorylation was observed in RD cells after treatment with IGFBP-6, whereas no change was seen in phospho-ERK1/2 levels. Phospho-JNK was not detected. IGFBP-6-induced cell migration was inhibited by SB203580, an inhibitor of p38 MAPK, and PD98059, an inhibitor of ERK1/2 MAPK activation. In contrast, SP600125, a JNK MAPK inhibitor, had no effect on migration. Knockdown of p38 MAPK using short interfering RNA blocked IGFBP-6-induced migration of RD cells. These results indicate that p38 MAPK is involved in IGFBP-6-induced IGF-independent RD cell migration.