Downregulation of hepatic stem cell factor by Vivo-Morpholino treatment inhibits mast cell migration and decreases biliary damage/senescence and liver fibrosis in Mdr2−/− mice

Primary sclerosing cholangitis (PSC) is characterized by increased mast cell (MC) infiltration, biliary damage and hepatic fibrosis. Cholangiocytes secrete stem cell factor (SCF), which is a chemoattractant for c-kit expressed on MCs. We aimed to determine if blocking SCF inhibits MC migration, biliary damage and hepatic fibrosis.MethodsFVB/NJ and Mdr2^−/− mice were treated with Mismatch or SCF Vivo-Morpholinos. We measured (i) SCF expression and secretion; (ii) hepatic damage; (iii) MC migration/activation and histamine signaling; (iv) ductular reaction and biliary senescence; and (v) hepatic fibrosis. In human PSC patients, SCF expression and secretion were measured. In vitro, cholangiocytes were evaluated for SCF expression and secretion. Biliary proliferation/senescence was measured in cholangiocytes pretreated with 0.1% BSA or the SCF inhibitor, ISK03. Cultured HSCs were stimulated with cholangiocyte supernatant and activation measured. MC migration was determined with cholangiocytes pretreated with BSA or ISK03 loaded into the bottom of Boyden chambers and MCs into top chamber.ResultsBiliary SCF expression and SCF serum levels increase in human PSC. Cholangiocytes, but not hepatocytes, from SCF Mismatch Mdr2^−/− mice have increased SCF expression and secretion. Inhibition of SCF in Mdr2^−/− mice reduced (i) hepatic damage; (ii) MC migration; (iii) histamine and SCF serum levels; and (iv) ductular reaction/biliary senescence/hepatic fibrosis. In vitro, cholangiocytes express and secrete SCF. Blocking biliary SCF decreased MC migration, biliary proliferation/senescence, and HSC activation.ConclusionCholangiocytes secrete increased levels of SCF inducing MC migration, contributing to biliary damage/hepatic fibrosis. Targeting MC infiltration may be an option to ameliorate PSC progression.     

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.     

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.     

The Notch2–Jagged1 interaction mediates stem cell factor signaling in erythropoiesis

Stem cell factor (SCF), the ligand for the c-kit receptor, is essential for the production of red blood cells during development and stress erythropoiesis. SCF promotes erythroblast proliferation and survival, while delaying erythroid differentiation through mechanisms that are largely unknown. In cultures of primary human differentiating erythroblasts, we found that SCF induces an increase in the expression of Notch2, a member of the Notch family implicated in the control of cell growth and differentiation. Functional inhibition of either Notch or its ligand Jagged1 inhibited the effects of SCF on erythroid cell expansion. SCF also induced the expression of Hes-1 and GATA-2, which may contribute to transduce Notch2 signals in response to SCF. Transduction of primary erythroid precursors with a dominant-negative Notch2 mutant inhibited both basal and SCF-mediated erythroblast expansion, and counteracted the effects of SCF on erythroblast differentiation. These findings provide a clue to understand the effects of increased proliferation and delayed differentiation elicited by SCF on the erythroid compartment and indicate Notch2 as a new player in the regulation of red cell differentiation.     

Stem cell factor/c-Kit interactions regulate human islet-epithelial cluster proliferation and differentiation

Stem cell factor (SCF), a progenitor cell growth factor, binds to and activates the c-Kit receptor tyrosine kinase, which is critical for early stem cell differentiation in haematopoiesis and gametogenesis. Nothing is known regarding these interactions during islet development in the human fetal pancreas. The present study was to investigate whether an increase in c-Kit receptor activity in isolated human fetal islet-epithelial clusters, by giving exogenous SCF, would promote beta-cell development. In the intact fetal pancreas, SCF and c-Kit were observed co-localizing with cytokeratin 19 in both ductal and newly forming islet cells. Islet cells isolated from 14 to 16 weeks fetal pancreata were cultured with SCF (50 ng/ml) or vehicle for 48 h. We observed an increase in the number of c-Kit-, pancreatic and duodenal homeobox gene 1- (PDX-1-), insulin- and glucagon-expressing cells in the SCF-treated group (PDX-1 and insulin, p < 0.05). PDX-1 and c-Kit mRNA levels were also up-regulated in the SCF group (PDX-1, p < 0.05), with no change in preproinsulin or proglucagon gene expression. Co-localization of insulin with PDX-1 or c-Kit was observed frequently in SCF-treated cultures. A significantly (p < 0.05) greater proliferative capacity of islet-epithelial clusters was found in the SCF group in parallel with increased (p < 0.02) phosphorylation of Akt in a phosphatidylinositol-3 kinase (PI3K)-dependent manner. Our results demonstrate that SCF/c-Kit interactions are likely to be involved in mediating islet cell differentiation and proliferation during human fetal pancreatic development, and that phosphorylated Akt may have a role downstream of SCF/c-Kit signaling.     

Induction of globin mRNA expression by interleukin-3 in a stem cell factor-dependent SV-40 T-antigen-immortalized multipotent hematopoietic cell line

Erythropoiesis requires the stepwise action on immature progenitors of several growth factors, including stem cell factor (SCF), interleukin 3 (IL-3), and erythropoietin (Epo). Epo is required to sustain proliferation and survival of committed progenitors and might further modulate the level of expression of several erythroid genes, including globin genes. Here we report a new SCF-dependent immortalized mouse progenitor cell line (GATA-1 ts SCF) that can also grow in either Epo or IL-3 as the sole growth factor. When grown in SCF, these cells show an "open" chromatin structure of the beta-globin LCR, but do not significantly express globin. However, Epo or IL-3 induce globin expression and are required for its maintainance. This effect of IL-3 is unexpected as IL-3 was previously reported either to be unable to induce hemoglobinization, or even to antagonize it. This suggests that GATA-1 ts SCF cells may have progressed to a stage in which globin genes are already poised for expression and only require signal(s) that can be elicited by either Epo or IL-3. Through the use of inhibitors, we suggest that p38 may be one of the molecules modulating induction and maintenance of globin expression.     

Cellular and subcellular calcium accumulation during glutamate-induced injury in cerebellar granule neurons

Abstract We have investigated the role of Ca2+ accumulation and neuronal injury in cerebellar granule neurons after glutamate receptor overactivation. After the removal of the free cytosolic Ca2+ we identified an extensive second Ca2+ fraction (SCF) that is retained within the neurons after glutamate receptor overactivation. The SCF reaches a plateau within 10 min with the magnitude of this SCF accumulation reflecting the extent of the neuronal injury that occurs within the neurons. The existence of this SCF is sensitive to both NMDA receptor antagonists and mitochondrial inhibitors but is unaffected by agents that deplete endoplasmic reticulum Ca2+, indicating that this Ca2+ fraction may be located within the mitochondria. Through the isolation of mitochondria from cerebellar granule neurons treated with glutamate we have shown that the majority of the SCF is mitochondrial in location. On the removal of the glutamate stimulus the SCF recovers at a slower rate than the free Ca2+ concentration within the neuron. This is intriguing, as it implies a capacity to remember previous excitatory events. Most significantly we have shown that a short pre-application of subthreshold glutamate or kainate blocks both SCF Ca2+ accumulation and extensive neuronal injury in response to high concentrations of glutamate. These findings may be relevant to the observations of pre-conditioning in the brain and heart.     

Stem Cell Factor Regulates Human Melanocyte-Matrix Interactions

Stem cell factor (SCF) is hypothesized to play a critical role in the migration of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, c-kit, result in defects in coat pigmentation in mice and in skin pigmentation in humans. In this report we directly show that SCF alters the adhesion and migration of human melanocytes to extracellular matrix (ECM) ligands and regulates integrin expression at the protein level. SCF decreased adhesion of neonatal and fetal cells to collagen IV, and increased attachment of fetal cells to laminin. Attachment of fetal cells to fibronectin was decreased, but was unchanged in neonatal cells. Flow cytometry analysis of neonatal melanocytes showed that SCF down-regulated the expression of the α2 receptor, and up-regulated the expression of the α3, α5 and β1 integrin receptors. SCF down-regulated expression of α2, α5 and β1 integrins by fetal melanocytes, and up-regulated expression of the αv and α3 integrin receptors. Analysis of melanocyte migration using time-lapse videomicroscopy showed that SCF significantly increased migration of neonatal, but not fetal, melanocytes on fibronectin (FN). We conclude that SCF regulates integrin expression at the protein level and that SCF has pleiotropic effects on melanocyte attachment and migration on ECM ligands. We suggest that this may be one mechanism by which SCF regulates melanocyte migration during development of the skin.     

Stem cell factor consistently improves thymopoiesis after experimental transplantation of murine or human hematopoietic stem cells in immunodeficient mice

Deficient thymopoiesis is a pivotal determinant of impaired immune competence following hematopoietic stem cell transplantation (HSCT). Stem cell factor (SCF) is essentially involved in early thymopoiesis. We evaluated whether SCF administration would improve recovery of thymopoiesis following HSCT in immunodeficient mice receiving: 1) bone marrow (BM) transplantation of congenic mice; or 2) human fetal liver HSCT in the human immune system mouse model. Following murine BM transplantation, SCF significantly enhanced thymopoiesis and peripheral T cell recovery in lymph nodes and spleen. SCF did not affect BM lymphoid progenitor recovery and/or expansion. Median thymic cellularity increased from 0.9 in PBS- to 266 × 10(4)/thymus in SCF-treated mice (p = 0.05). Following human HSCT in human immune system mice, higher thymic cellularity was observed in SCF-treated mice. Double-negative and early double-positive thymocyte subsets increased, but especially late double-positive, CD4 single-positive, and CD8 single-positive thymocyte subsets were significantly enhanced (p < 0.05). These results show that exogenous supply of SCF may significantly improve murine and human posttransplant thymopoiesis, for which the effect is probably exerted by directly promoting T cell development intrathymically rather than by enhanced entry of prethymically expanded lymphoid progenitors.     

Synergistic growth of stem cell factor and granulocyte macrophage colony-stimulating factor involves kinase-dependent and -independent contributions from c-Kit

Stem cell factor (SCF) binds and activates the receptor tyrosine kinase c-Kit, and this interaction is critical for normal hematopoiesis. SCF also synergizes with a variety of growth factors, including those binding members of the cytokine receptor superfamily. The mechanisms mediating this synergy remain to be defined. The present study investigates both structural and biochemical cross-talk between c-Kit and the receptor for granulocyte macrophage colony-stimulating factor (GM-CSF). We have found that c-Kit forms a complex with the beta-chain of the GM-CSF receptor, and this interaction involves the first part of the c-Kit kinase domain. Although inhibition of c-Kit kinase activity completely blocked SCF-induced proliferation, there was still greater than additive growth induced by SCF in combination with GM-CSF. In contrast, an inhibitory antibody against the extracellular domain of c-Kit (K-27) completely inhibited growth in response to SCF alone or in combination with GM-CSF. These results support a kinase-independent component of the synergistic growth induced by SCF and GM-CSF that may relate to interaction of these receptors. It is also clear that a significant part of the synergistic growth is dependent of c-Kit kinase activity. Although synergistic increases in phosphorylation of c-Kit and the beta-chain of the GM-CSF receptor were not observed, SCF and GM-CSF in combination prolonged the duration of Erk1/2 phosphorylation in a phosphatidylinositol 3-kinase-dependent manner. Consistent with these findings, phosphatidylinositol 3-kinase is synergistically activated by SCF and GM-CSF together. Hence, c-Kit makes both kinase-independent and -dependent contributions to the proliferative synergy induced by SCF in combination with GM-CSF.     

Application of self-cycling fermentation technique to the production of poly-beta-hydroxybutyrate

The production of poly-beta-hydroxybutyrate (PHB) by Alcaligenes eutrophus DSM 545 in a cyclone bioreactor was compared using various culture methods: batch, fed-batch, and self-cycling fermentation (SCF) with and without extended periods of nutrient deprivation. SCF is a semi-continuous method that results in a nutrient limitation for every successive generation of cells and, therefore, may have advantages for products whose formation follow secondary metabolite kinetics. Use of the SCF technique without extended nutrient deprivation produced a PHB concentration of 1.2 g L(-1) as 40% of the biomass dry weight. With nitrogen deprivation for 4 or 6 h, the concentration of PHB decreased when compared to the standard SCF technique. However, nitrogen deprivation periods of 8 h resulted in an increase in PHB concentration to 2.7 g L(-1) or 59% of the biomass dry weight. The nutrient cycling may act to repress PHB accumulation during periods of nitrogen deprivation, unless a time threshold has been reached, after which PHB accumulation occurs as in normal batch culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 815-820, 1997.     

Development of Melanocyte Progenitors in Murine Steel Mutant Neural Crest Explants Cultured With Stem Cell Factor,Endothelin-3, or TPA

Stem cell factor (SCF) has been suggested to be indispensable for the development of neural crest cells into melanocytes because Steel mutant mice (i.e., Sl/Sf¹) have no pig-mented hairs. On the other hand, it has been demonstrated that the addition of endothelin 3 (ET-3) or TPA to neural crest cell cultures can induce melanocyte differentiation without addition of extrinsic SCF. In this study, we excluded the influence of intrinsic SCF by using SI/SI mouse embryos to study more precisely the effects of natural cytokines, such as extrinsic soluble SCF or ET-3, or chemical reagents, such as TPA or cholera toxin. We found that SCF is supplied within the wild-type neural crest explants and that ET-3 cannot induce melanocyte differentiation or proliferation without SCF. These results indicate that SCF plays a critical role in survival or G1/S entry of melanocyte progenitors and that SCF initially stimulates their proliferation and then ET-3 accelerates their proliferation and differentiation. TPA has the ability to elicit neural crest cell differentiation into melanocytes without exogenously added SCF but it is not as effective as SCF because many more melanocytes developed in the wild-type neural crest explants cultured with TPA.     

Tec kinase associates with c-kit and is tyrosine phosphorylated and activated following stem cell factor binding.

Stem cell factor (SCF) plays a crucial role in hematopoiesis through its interaction with the receptor tyrosine kinase c-kit. However, the signaling events that are activated by this interaction and involved in the control of growth or differentiation are not completely understood. We demonstrate here that Tec, a cytoplasmic, src-related kinase, physically associates with c-kit through a region that contains a proline-rich motif, amino terminal of the SH3 domain. Following SCF binding, Tec is tyrosine phosphorylated and its in vitro kinase activity is increased. Tyrosine phosphorylation of Tec is not detected in the response to other cytokines controlling hematopoiesis, including colony-stimulating factor-1 (CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3). Conversely, the cytoplasmic kinase JAK2 is activated by IL-3 but not by SCF stimulation. The activation of distinct cytoplasmic kinases may account for the synergy seen in the actions of SCF and IL-3 on hematopoietic stem cells.     

Modification of expression of stem cell factor by various cytokines.

The local production of stem cell factor (SCF) may be an important mechanism for regulating proliferation, differentiation, and migration of various cells bearing c-kit receptors, and might be susceptible to the cytokines that serve in inflammation and tissue repair. We have demonstrated that in three murine cell lines, Balb/3T3A31, MC3T3-E1, and C3H-2K, which constitutively produced SCF with different quantity, the SCF mRNA expression was greatly enhanced in response to basic fibroblast growth factor (bFGF) or transforming growth factor beta1 (TGF-beta1). The study was carried out by in situ hybridization utilizing nonradioactive oligonucleotide probes and quantitative image analysis. Leukemia inhibitory factor (LIF) or interleukin-4 (IL-4) moderately increased SCF mRNA in all cell lines, but IL-3 did not. The dot-blot enzyme-linked immunosorbent assay (ELISA) further confirmed that SCF protein production in these cell lines and bone marrow stromal cells was markedly enhanced by TGF-beta1, although TGF-beta1 suppressed the proliferation of all these cells. bFGF also enhanced the SCF production in these cell lines, but did not in bone marrow stromal cells, suggesting a difference in their susceptibility to the cytokine. Our results suggest that TGF-beta1 and bFGF potentially modulate the biological function of cells bearing c-kit receptors through the modulation of SCF production in fibroblasts.     

Role of SKP1-CUL1-F-Box-Protein (SCF) E3 Ubiquitin Ligases in Skin Cancer

Many biological processes such as cell proliferation, differentiation, and cell death depend precisely on the timely synthesis anddegradation of key regulatory proteins. While protein synthesis can be regulated at multiple levels, protein degradation is mainlycontrolled by the ubiquitineproteasome system (UPS), which consists of two distinct steps: (1) ubiquitylation of targeted protein by E1ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase, and (2) subsequent degradation by the 26Sproteasome. Among all E3 ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) E3 ligases are the largest family and are responsiblefor the turnover of many key regulatory proteins. Aberrant regulation of SCF E3 ligases is associated with various human diseases, such ascancers, including skin cancer. In this review, we provide a comprehensive overview of all currently published data to define a promotingrole of SCF E3 ligases in the development of skin cancer. The future directions in this area of research are also discussed with an ultimategoal to develop small molecule inhibitors of SCF E3 ligases as a novel approach for the treatment of human skin cancer. Furthermore,altered components or substrates of SCF E3 ligases may also be developed as the biomarkers for early diagnosis or predicting prognosis.     

NIPA defines an SCF-type mammalian E3 ligase that regulates mitotic entry

The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-box subunit of the SCF specifically recruiting a given substrate to the SCF core. Here we identify NIPA (nuclear interaction partner of ALK) as a human F-box-containing protein that defines an SCF-type E3 ligase (SCF(NIPA)) controlling mitotic entry. Assembly of this SCF complex is regulated by cell-cycle-dependent phosphorylation of NIPA, which restricts substrate ubiquitination activity to interphase. We show nuclear cyclin B1 to be a substrate of SCF(NIPA). Inactivation of NIPA by RNAi results in nuclear accumulation of cyclin B1 in interphase, activation of cyclin B1-Cdk1 kinase activity, and premature mitotic entry. Thus, SCF(NIPA)-based ubiquitination may regulate S-phase completion and mitotic entry in the mammalian cell cycle.     

Stem cell factor is localized in, released from, and cleaved by human mast cells.

Stem cell factor (SCF) is the most important cytokine regulating human mast cell growth and functions. The immunogold technique showed SCF in the secretory granules of skin mast cells and in lung parenchymal mast cells (HLMC). Immunoreactive SCF (iSCF) was detected in cell lysates of HLMC, but not in basophils; iSCF and histamine were detected in supernatants of HLMC 3 min after challenge with anti-FcepsilonRI or anti-IgE, and iSCF in supernatants rapidly declined after 30 min, whereas histamine remained unchanged for 120 min. HPLC and electrospray mass spectrometry (ES/MS) analysis of recombinant human SCF1-166 (18,656. 9 +/- 0.9 Da) treated with chymase showed a polypeptide of 17,977.1 +/- 0.6 Da and a minor component of 697.4 +/- 0.1 Da generated by specific cleavage at Phe159. SCF1-166 and SCF1-159 similarly activated HLMC, potentiated anti-IgE-induced activation of these cells, and stimulated HLMC chemotaxis. SCF159-166 had no effect on mast cells. Western blot analysis of supernatants of anti-IgE-activated HLMC incubated with recombinant human SCF1-166 showed that SCF1-166 was rapidly cleaved to SCF1-159 and SCF1-144. Experiments with supernatants of anti-IgE-activated HLMC incubated with SCF1-166 yielded similar results. In conclusion, SCF is stored in mast cell secretory granules and is immunologically released by human mast cells. SCF1-166 is rapidly and specifically cleaved to SCF1-159 by chymase, which retains its biological effect on mast cells. SCF is also cleaved by other proteases to several SCF species whose possible biological activities remain to be established.