RESUMEN
BACKGROUND: Incidences of inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, are escalating worldwide and can be considered a global public health problem. Given that the gold standard approach to IBD therapeutics focuses on reducing the severity of symptoms, there is an urgent unmet need to develop alternative therapies that halt not only inflammatory processes but also promote mucosal repair. Previous studies have identified increased stem cell factor (SCF) expression in inflamed intestinal mucosal tissues. However, the role that SCF plays in mediating intestinal inflammation and repair has not been explored. METHODS: Changes in the expression of SCF were evaluated in the colonic tissue of healthy mice and during dextran sodium sulfate (DSS)-induced colitis. Furthermore, mucosal wound healing and colitis severity were analyzed in mice subjected to either mechanical biopsy or DSS treatment, respectively, following intestinal epithelial cell-specific deletion of SCF or anti-SCF antibody administration. RESULTS: We report robust expression of SCF by intestinal epithelial cells during intestinal homeostasis with a switch to immune cell-produced SCF during colitis. Data from mice with intestinal epithelial cell-specific deletion of SCF highlight the importance of immune cell-produced SCF in driving the pathogenesis of colitis. Importantly, antibody-mediated neutralization of total SCF or the specific SCF248 isoform decreased immune cell infiltration and enhanced mucosal wound repair following biopsy-induced colonic injury or DSS-induced colitis. CONCLUSIONS: These data demonstrate that SCF functions as a pro-inflammatory mediator in mucosal tissues and that specific neutralization of SCF248 could be a viable therapeutic option to reduce intestinal inflammation and promote mucosal wound repair in individuals with IBD.
Our investigation demonstrates that blocking cleavable SCF248 isoform by administration of specific stem cell factor antibodies enhances healing of the intestinal mucosa and restores critical barrier function, suggesting an alternative therapeutic option to treat individuals with active IBD.
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Colitis Ulcerosa , Colitis , Enfermedades Inflamatorias del Intestino , Animales , Ratones , Colitis/tratamiento farmacológico , Colitis/patología , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Inflamación/tratamiento farmacológico , Inflamación/patología , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/patología , Factor de Células Madre/antagonistas & inhibidores , Factor de Células Madre/metabolismoRESUMEN
Food allergy is a growing public health problem with ~15 million people affected in the United States. In allergic food disease, IgE on mast cells bind to ingested antigens leading to the activation and degranulation of mast cells. Stem cell factor (SCF) is mast cell growth and activation factor that is required for peripheral tissue mast cells. We targeted a specific isoform of SCF, the larger 248 amino acid form, that drives peripheral tissue mast cell differentiation using a specific monoclonal antibody in a model of food allergy. Ovalbumin sensitized and intragastrically challenged mice were monitored for symptoms of anaphylaxis including respiratory distress, diarrhea, and a reduction in body temperature. During the second week of challenges, allergic mice were injected with an antibody to block SCF248 or given IgG control. Mice treated with α-SCF248 had a decreased incidence of diarrhea and no reduction in body temperature suggesting a reduction in anaphylaxis compared to IgG control treated animals. Re-stimulated mesenteric lymph nodes indicated that α-SCF248 treated mice had decreased OVA-specific Th2 cytokine production compared to IgG control treated allergic animals. The reduction of food induced anaphylaxis was accompanied by a significant reduction in gut leak. The mesenteric lymph node cells were analyzed by flow cytometry and showed a decrease in the number of type 2 innate lymphoid cells in mice injected with α-SCF248. Morphometric enumeration of esterase+ mast cells demonstrated a significant reduction throughout the small intestine. Using a more chronic model of persistent food-induced anaphylaxis, short term therapeutic treatment with α-SCF248 during established disease effectively blocked food induced anaphylaxis. Together, these data suggest that therapeutically blocking SCF248 in food allergic animals can reduce the severity of food allergy by reducing mast cell mediated disease activation.
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Anafilaxia/inmunología , Anafilaxia/prevención & control , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Neutralizantes/farmacología , Hipersensibilidad a los Alimentos/inmunología , Factor de Células Madre/antagonistas & inhibidores , Alérgenos/inmunología , Anafilaxia/diagnóstico , Anafilaxia/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Biomarcadores , Biopsia , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Femenino , Hipersensibilidad a los Alimentos/diagnóstico , Hipersensibilidad a los Alimentos/tratamiento farmacológico , Inmunoglobulina E/inmunología , Inmunofenotipificación , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Mastocitos/metabolismo , Ratones , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
In mammals, progressive activation of primordial follicles is essential for maintenance of the reproductive lifespan. Several reports have demonstrated that mitogen-activated protein kinases 3 and 1 (MAPK3/1)-mammalian target of rapamycin complex 1 (mTORC1) signaling in pre-granulosa cells promotes primordial follicle activation by increasing KIT ligand (KITL) expression and then stimulating phosphatidylinositol 3 kinase signaling in oocytes. However, the mechanism of mTORC1 signaling in the promotion of KITL expression is unclear. Immunofluorescence staining results showed that phosphorylated cyclic AMP response element-binding protein (CREB) was mainly expressed in pre-granulosa cells. The CREB inhibitor KG-501 and CREB knockdown by Creb siRNA significantly suppressed primordial follicle activation, reduced pre-granulosa cell proliferation and dramatically increased oocyte apoptosis. Western blotting results demonstrated that both the MAPK3/1 inhibitor U0126 and mTORC1 inhibitor rapamycin significantly decreased the levels of phosphorylated CREB, indicating that MAPK3/1-mTORC1 signaling is required for CREB activation. Furthermore, CREB could bind to the Kitl promoter region, and KG-501 significantly decreased the expression levels of KITL. In addition, KG-501 and CREB knockdown significantly decreased the levels of phosphorylated Akt, leading to a reduced number of oocytes with Foxo3a nuclear export. KG-501 also inhibited bpV (HOpic)-stimulated primordial follicle activation. Taken together, the results show that CREB is required for MAPK3/1-mTORC1 signaling-promoted KITL expression followed by the activation of primordial follicles.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Folículo Ovárico/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Ratones , Ratones Endogámicos ICR , Naftoles/farmacología , Organofosfatos/farmacología , Folículo Ovárico/efectos de los fármacos , Fosforilación , Transducción de Señal/genética , Factor de Células Madre/antagonistas & inhibidores , Factor de Células Madre/metabolismo , Técnicas de Cultivo de Tejidos , Compuestos de Vanadio/antagonistas & inhibidores , Compuestos de Vanadio/farmacologíaRESUMEN
PURPOSE: Bladder smooth muscle cell death accompanied by hyperplasia and hypertrophy, as induced by inflammation, is the primary cause for poor bladder function. There are emerging evidences on the role of chronic inflammation as a factor involved in carcinogenesis and progression. We aim to determine the bladder smooth muscle pathological changes and dysfunction in chronic prostatitis (CP), to investigate whether resveratrol can improve the urinary dysfunction and the role of c-kit/SCF pathway, that has been associated with the smooth muscle carcinogenesis. METHOD: Rat model of CP was established via subcutaneous injections of DPT vaccine and subsequently treated with resveratrol. H&E staining was performed to identify the histopathological changes in prostates and bladders. Western blotting and immunohistochemical staining examined the expression level of C-kit, stem cell factor (SCF), Sirt1, apoptosis associated proteins. RESULTS: the model group exhibited severe diffuse chronic inflammation, characterized by leukocyte infiltration and papillary frond protrusion into the gland cavities, and a notable increase in prostatic epithelial height. Meanwhile, bladder muscle arranged in disorder with fracture, and cells appeared atypia. The activity of C-kit/SCF was up-regulated, the carcinogenesis associated proteins are dysregulated significantly in CP rats. Resveratrol treatment significantly improved these factors by Sirt1 activation. CONCLUSIONS: activated c-kit/SCF and bladder muscle carcinogenesis were involved in the pathological processes of CP, which was improved after resveratrol treatment via the downregulation of c-kit/SCF by activating Sirt1.
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Carcinogénesis/metabolismo , Músculo Liso/metabolismo , Prostatitis/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Sirtuina 1/metabolismo , Factor de Células Madre/metabolismo , Estilbenos/uso terapéutico , Animales , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/uso terapéutico , Carcinogénesis/efectos de los fármacos , Carcinogénesis/patología , Enfermedad Crónica , Progresión de la Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Masculino , Músculo Liso/efectos de los fármacos , Músculo Liso/patología , Prostatitis/tratamiento farmacológico , Prostatitis/patología , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Ratas , Resveratrol , Factor de Células Madre/antagonistas & inhibidores , Estilbenos/farmacología , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/metabolismo , Vejiga Urinaria/patologíaRESUMEN
BACKGROUND: Sorafenib is a multi-kinase inhibitor for treating advanced hepatocellular and renal cell carcinomas by targeting various types of receptors and signaling molecules, including vascular endothelial growth factor receptors, platelet-derived growth factor receptor, and Raf-1. Sorafenib may cause diverse cutaneous adverse reactions, including hand-foot reaction, facial and scalp eruptions, alopecia and pruritus. However, the mechanism of these adverse effects has not been well-investigated. OBJECTIVE: Mast cells (MCs) are reported to be associated with various types of skin diseases. To investigate the mechanism of sorafenib-induced cutaneous adverse effects, we focused on MCs in situ. METHODS: We evaluated skin samples of organ cultured normal human skin treated with sorafenib using c-Kit, tryptase, and stem cell factor (SCF), Ki-67, and TUNEL immunohistochemistry as well as quantitative real-time polymerase chain reaction to evaluate MC number, degranulation, proliferation, and apoptosis in situ. RESULTS: Sorafenib significantly increased the number and degranulation of skin-type MCs compared with the vehicle-treated control group in situ. However, sorafenib did not affect MC proliferation and apoptosis, suggesting that it stimulated MC maturation from resident precursors. Furthermore, sorafenib increased SCF expression in situ. The increase in MC number by sorafenib was abrogated by co-administration of SCF neutralizing antibody or the phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, but not the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor, PD98059. This suggests that SCF is involved in sorafenib-induced MC maturation. In addition, the compensatory upregulation of PI3K-signaling from inhibition of MAPK signaling by sorafenib might stimulate MC maturation in situ. We also evaluated MCs within the skin samples from patients with drug eruptions by sorafenib administration. The total and degranuated MCs number as well as SCF expression was significantly increased compared to healthy individuals. CONCLUSION: Our results contribute to a better understanding of the mechanism by which sorafenib induces adverse cutaneous reactions via activation of skin-type MC degranulation and maturation. This activation appears to be related to PI3K signaling and SCF production, which could be a new targets for treating sorafenib-induced adverse reactions.
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Degranulación de la Célula/efectos de los fármacos , Erupciones por Medicamentos/patología , Mastocitos/efectos de los fármacos , Niacinamida/análogos & derivados , Compuestos de Fenilurea/efectos adversos , Inhibidores de Proteínas Quinasas/efectos adversos , Piel/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Androstadienos/farmacología , Antineoplásicos/efectos adversos , Apoptosis/efectos de los fármacos , Biopsia , Recuento de Células , Proliferación Celular/efectos de los fármacos , Erupciones por Medicamentos/etiología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Flavonoides/farmacología , Humanos , Masculino , Mastocitos/fisiología , Persona de Mediana Edad , Niacinamida/efectos adversos , Técnicas de Cultivo de Órganos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Transducción de Señal/efectos de los fármacos , Piel/citología , Piel/metabolismo , Piel/patología , Sorafenib , Factor de Células Madre/antagonistas & inhibidores , Factor de Células Madre/metabolismo , Factor A de Crecimiento Endotelial Vascular , Wortmanina , Adulto JovenRESUMEN
Imatinib mesylate is an anti-cancer agent that competitively inhibits several receptor tyrosine kinases (RTKs). RTKs play important roles in the regulation of primordial follicle formation, the recruitment of primordial follicles into the pool of growing follicles and maturation of the follicles. In the present study, we investigated the effects of the tyrosine kinase inhibitor imatinib on primordial follicle assembly and early folliculogenesis in postnatal rats. Female Sprague-Dawley rats were treated with either imatinib (150mg/kg) or placebo (water) on postnatal days 2-4. Bilateral ovariectomy was performed on postnatal day 2 and 5. Histology, immunohistochemistry, and mRNA analysis were performed. Imatinib treatment was associated with increased density of the multi-oocyte follicles (P<0.01), oogonia (p<0.01) and germline clusters (P<0.05), decreased activation of primordial follicles, increased expression of c-Kit and AMH, and decreased protein expression of Kit-ligand and GDF9 when compared to age-matched controls. In conclusion, imatinib affects folliculogenesis in postnatal rat ovaries by delaying the cluster breakdown, follicular assembly and early activation of the primordial follicle pool.
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Antineoplásicos/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Mesilato de Imatinib/farmacología , Oogénesis/efectos de los fármacos , Células Madre Oogoniales/efectos de los fármacos , Folículo Ovárico/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Animales , Animales Recién Nacidos , Hormona Antimülleriana/química , Hormona Antimülleriana/genética , Hormona Antimülleriana/metabolismo , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Femenino , Factor 9 de Diferenciación de Crecimiento/antagonistas & inhibidores , Factor 9 de Diferenciación de Crecimiento/genética , Factor 9 de Diferenciación de Crecimiento/metabolismo , Inmunohistoquímica , Oogonios/citología , Oogonios/efectos de los fármacos , Oogonios/metabolismo , Células Madre Oogoniales/citología , Folículo Ovárico/citología , Folículo Ovárico/crecimiento & desarrollo , Proteínas Proto-Oncogénicas c-kit/agonistas , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Factor de Células Madre/antagonistas & inhibidores , Factor de Células Madre/genética , Factor de Células Madre/metabolismoRESUMEN
Stem cell factor (SCF) plays an important role in tumor growth and metastasis. However, the function of SCF in regulating stemness and migration of cancer stem cells (CSCs) remains largely undefined. Here, we report that non-adhesive culture system can enrich and expand CSCs derived from Lewis lung carcinoma (LLC) cells and that the expression level of SCF in CSCs was higher than those in LLC cells. Silencing SCF via short hairpin (sh) RNA lentivirus transduction attenuated sphere formation and inhibited expressions of stemness genes, ALDH1, Sox2, and Oct4 of CSCs in vitro and in vivo. Moreover, SCF-silenced CSCs inhibited the migration and epithelial-mesenchymal transition, with decreased expression of N-cadherin, Vimentin, and increased expression of E-cadherin in vitro and in vivo. Finally, SCF-short hairpin RNA (shRNA) lentivirus transduction suppressed tumorigenicity of CSCs. Taken together, our findings unraveled an important role of SCF in CSCs derived from LLC cells. SCF might serve as a novel target for lung cancer therapy.
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Carcinoma Pulmonar de Lewis/patología , Movimiento Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal , Silenciador del Gen , Células Madre Neoplásicas/patología , Factor de Células Madre/metabolismo , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adipocitos/patología , Familia de Aldehído Deshidrogenasa 1 , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/metabolismo , Western Blotting , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/metabolismo , Diferenciación Celular , Proliferación Celular/efectos de los fármacos , Femenino , Técnica del Anticuerpo Fluorescente , Técnicas para Inmunoenzimas , Isoenzimas/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , ARN Interferente Pequeño/genética , Retinal-Deshidrogenasa/metabolismo , Factor de Células Madre/antagonistas & inhibidores , Factor de Células Madre/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The development of prostate cancer (PCa) is intimately associated with the hormonal environment, and the sex steroids estrogens have been implicated in prostate malignancy. However, if some studies identified estrogens as causative agents of PCa, others indicated that these steroids have a protective role counteracting prostate overgrowth. The tyrosine kinase receptor c-KIT and its ligand, the stem cell factor (SCF), have been associated with the control of cell proliferation/apoptosis and prostate carcinogenesis, and studies show that estrogens regulate their expression in different tissues, though, in the case of prostate this remains unknown. The present study aims to evaluate the role of 17ß-estradiol (E2) in regulating the expression of SCF/c-KIT in human prostate cell lines and rat prostate, and to investigate the consequent effects on prostate cell proliferation and apoptosis. qPCR, Western Blot, and immuno(cito)histochemistry analysis showed that E2-treatment decreased the expression of SCF and c-KIT both in human prostate cells and rat prostate. Furthermore, the diminished expression of SCF/c-KIT was underpinned by the diminished prostate weight and reduced proliferation index. On the other hand, the results of TUNEL labelling, the increased activity of caspase-3, and the augmented expression of caspase-8 and Fas system in the prostate of E2-treated animals indicated augmented apoptosis in response to E2. The obtained results demonstrated that E2 down-regulated the expression of SCF/c-KIT system in prostate cells, which was associated with antiproliferative and proapoptotic effects. Moreover, these findings support the protective role of estrogens in PCa and open new perspectives on the application of estrogen-based therapies.
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Apoptosis/fisiología , Proliferación Celular/fisiología , Estrógenos/farmacología , Próstata/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Células Madre/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Humanos , Masculino , Próstata/citología , Próstata/efectos de los fármacos , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Ratas , Ratas Wistar , Factor de Células Madre/antagonistas & inhibidoresRESUMEN
BACKGROUND & AIMS: Colon tumors contain a fraction of undifferentiated stem cell-like cancer cells with high tumorigenic potential. Little is known about the signals that maintain these stem-like cells. We investigated whether differentiated tumor cells provide support. METHODS: We established undifferentiated colonosphere cultures from human colon tumors and used them to generate stably differentiated cell lines. Antibody arrays were used to identify secreted factors. Expression of genes involved in stemness, differentiation, and the epithelial to mesenchymal transition was measured using reverse transcription quantitative polymerase chain reaction. Expression of KIT in human tumors was analyzed with gene expression arrays and by immunohistochemistry. Colonospheres were injected into the livers of CBy.Cg-Foxn1nu/J mice. After liver tumors had formed, hypoxia was induced by vascular clamping. RESULTS: Differentiated cells from various tumors, or medium conditioned by them, increased the clonogenic capacity of colonospheres. Stem cell factor (SCF) was secreted by differentiated tumor cells and supported the clonogenic capacity of KIT(+) colonosphere cells. Differentiated tumor cells induced the epithelial to mesenchymal transition in colonosperes; this was prevented by inhibition of KIT or SCF. SCF prevented loss of clonogenic potential under differentiation-inducing conditions. Suppression of SCF or KIT signaling greatly reduced the expression of genes that regulate stemness and the epithelial to mesenchymal transition and inhibited clonogenicity and tumor initiation. Bioinformatic and immunohistochemical analyses revealed a correlation between expression of KIT- and hypoxia-related genes in colon tumors, which was highest in relapse-prone mesenchymal-type tumors. Hypoxia induced expression of KIT in cultured cells and in human colon tumor xenografts and this contributed to the clonogenic capacity of the tumor cells. CONCLUSIONS: Paracrine signaling from SCF to KIT, between differentiated tumor cells and undifferentiated stem-like tumor cells, helps maintain the stem-like features of tumor cells, predominantly under conditions of hypoxia.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Diferenciación Celular , Neoplasias del Colon/enzimología , Células Madre Neoplásicas/enzimología , Comunicación Paracrina , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Células Madre/metabolismo , Animales , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Hipoxia de la Célula , Proliferación Celular , Técnicas de Cocultivo , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Desnudos , Células Madre Neoplásicas/patología , Comunicación Paracrina/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-kit/genética , Transducción de Señal , Esferoides Celulares , Factor de Células Madre/antagonistas & inhibidores , Factor de Células Madre/genética , Factores de Tiempo , Carga Tumoral , Células Tumorales CultivadasRESUMEN
The fine balance between proliferation and differentiation of vascular smooth muscle cells (VSMCs) is indispensable for the maintenance of healthy blood vessels, whereas an increase in proliferation participates in pathologic cardiovascular events such as atherosclerosis and restenosis. Here we report that microRNA-34c (miR-34c) targets stem cell factor (SCF) to inhibit VSMC proliferation and neointimal hyperplasia. In an animal model, miR-34c was significantly increased in the rat carotid artery after catheter injury. Transient transfection of miR-34c to either VSMCs or A10 cells inhibited cell survival by inducing apoptosis, which was accompanied by an increase in expression of p21, p27, and Bax. Transfection of miR-34c also attenuated VSMC migration. Bioinformatics showed that SCF is a target candidate of miR-34c. miR-34c down-regulated luciferase activity driven by a vector containing the 3'-untranslated region of SCF in a sequence-specific manner. Forced expression of SCF in A10 cells induced proliferation and migration, whereas knocking-down of SCF reduced cell survival and migration. miR-34c antagomir-induced VSMC proliferation was blocked by SCF siRNA. Delivery of miR-34c to rat carotid artery attenuated the expression of SCF and blocked neointimal hyperplasia. These results suggest that miR-34c is a new modulator of VSMC proliferation and that it inhibits neointima formation by regulating SCF.
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Arterias Carótidas/patología , MicroARNs/metabolismo , Factor de Células Madre/metabolismo , Regiones no Traducidas 3' , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Secuencia de Bases , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Hiperplasia , Masculino , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Neointima , Oligonucleótidos Antisentido/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Alineación de Secuencia , Factor de Células Madre/antagonistas & inhibidores , Factor de Células Madre/genéticaRESUMEN
Nobiletin is a unique flavonoid having polymethoxy groups and has exhibited anti-inflammatory and antiobesity effects. Here, we examined the inhibition of nobiletin on melanogenesis induced by endothelin-1 (ET) and stem cell factor (SCF) in normal human melanocytes. Nobiletin dose dependently reduced ET plus SCF-stimulated tyrosinase activity without causing cytotoxicity. Nobiletin reduced cAMP-response element-binding protein (CREB) phosphorylation and microphthalmia-associated transcription factor (MITF) expression, which is a key transcription factor for tyrosinase expression in pigmentation induced by ET plus SCF stimulation. Nobiletin treatment effectively decreased ET plus SCF-induced Raf-1, MEK and ERK1/2 phosphorylation and also downregulated the forskolin-induced phosphorylation of CREB. Furthermore, nobiletin inhibited ET plus SCF-triggered production of melanin and expression of MITF/tyrosinase in a three-dimensional human epidermal model. In accordance with protein expression, the expression of genes related to the pigmentation was also increased in the cells stimulated with ET plus SCF and the cotreatment with nobiletin decreased obviously the ET plus SCF-triggered gene expressions of tyrosinase, PMEL, TRP1 and MITF. Nobiletin contributes to hypopigmentation by downregulating MITF and tyrosinase expression through reduced Raf-1 phosphorylation. Our findings implicate nobiletin as a potential new whitening agent.
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Endotelina-1/antagonistas & inhibidores , Flavonas/farmacología , Melaninas/antagonistas & inhibidores , Melanocitos/efectos de los fármacos , Preparaciones para Aclaramiento de la Piel/farmacología , Factor de Células Madre/antagonistas & inhibidores , Células Cultivadas , Colforsina/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Endotelina-1/farmacología , Regulación de la Expresión Génica , Humanos , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Melaninas/biosíntesis , Melanocitos/citología , Melanocitos/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de Señal , Factor de Células Madre/farmacología , Técnicas de Cultivo de Tejidos , Antígeno gp100 del Melanoma/genética , Antígeno gp100 del Melanoma/metabolismoRESUMEN
We performed an immunohistochemical (IHC) study to determine the follicular expression of growth differentiation factor 9 (GDF-9), anti-Müllerian hormone (AMH), Kit Ligand (KL), and c-Kit in squirrel monkey ovary. Ovarian tissue fragments from 4 squirrel monkeys were collected by laparotomy and processed for classical histology and IHC. Additionally, follicle development was assessed by Ki67 immunostaining to evaluate proliferative status of granulosa cells. A total of 4025 follicles were examined (1475 for classical histology and 2550 for immunohistochemistry). More than 80% of the evaluated follicles were morphologically normal. The GDF-9 protein was detectable in oocyte cytoplasm from primordial (100%), primary (99.1%), and secondary (100%) follicles. The AMH was not expressed in primordial follicles but just in few primary follicles (13.8%). On the other hand, it was highly expressed in granulosa cells from secondary follicles (67.9%). c-Kit, KL receptor, was found in the oolemma of primordial (100%), primary (100%), and secondary (100%) follicles. The KL expression was observed in oocytes and granulosa cells from primordial (94.9%), primary (91.6%) and secondary follicles (100%). Ki67 immunostaining was observed in granulosa cells from primary (5.7%) and secondary (54.8%) follicles but not in primordial follicles. In conclusion, we described the localization of GDF-9, KL, c-Kit, and Ki67 proteins and confirmed the presence of AMH protein in preantral follicles from squirrel monkey. Our results offer contribution for understanding of folliculogenesis in neotropical nonhuman primates. Moreover, these markers can be used to assess follicular viability and functionality after cryopreservation, transplantation, or in vitro culture of ovarian tissue.
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Hormona Antimülleriana/análisis , Proliferación Celular , Factor 9 de Diferenciación de Crecimiento/análisis , Inmunohistoquímica , Folículo Ovárico/química , Proteínas Proto-Oncogénicas c-kit/análisis , Saimiri/fisiología , Factor de Células Madre/antagonistas & inhibidores , Factores de Edad , Animales , Femenino , Folículo Ovárico/citología , Saimiri/metabolismoRESUMEN
MiR-34c is considered a potent tumour suppressor because of its negative regulation of multiple target mRNAs that are critically associated with tumorigenesis and metastasis. In the present study, we demonstrated a novel target of miR-34c, KITLG, which has been implicated in colorectal cancer (CRC). First, we found a significant negative relationship between miR-34c and KITLG mRNA expression levels in CRC cell lines, including HT-29, HCT-116, SW480 and SW620 CRC cell lines. In silico analysis predicted putative binding sites for miR-34c in the 3' untranslated region (3'UTR) of KITLG mRNA. A dual-luciferase reporter assay further confirmed that KITLG is a direct target of miR-34c. Then, the cell lines were infected with lentiviruses expressing miR-34c or a miR-34c specific inhibitor. Restoration of miR-34c dramatically reduced the expression of KITLG mRNA and protein, while silencing of endogenous miR-34c increased the expression of KITLG protein. The miR-34c-mediated down-regulation of KITLG was associated with the suppression on proliferation, cellular transformation, migration and invasion of CRC cells, as well as the promotion on apoptosis. Knockdown of KITLG by its specific siRNA confirmed a critical role of KITLG down-regulation for the tumour-suppressive effects of miR-34c in CRC cells. In conclusion, our results demonstrated that miR-34c might interfere with KITLG-related CRC and could be a novel molecular target for CRC patients.
Asunto(s)
Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/prevención & control , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Factor de Células Madre/metabolismo , Apoptosis , Western Blotting , Ciclo Celular , Neoplasias Colorrectales/patología , Técnica del Anticuerpo Fluorescente , Humanos , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Células Madre/antagonistas & inhibidores , Factor de Células Madre/genética , Células Tumorales CultivadasRESUMEN
c-Kit and its ligand stem cell factor (SCF) are important for ß-cell survival and maturation; meanwhile, interactions between the Fas receptor (Fas) and Fas ligand are capable of triggering ß-cell apoptosis. Disruption of c-Kit signaling leads to severe loss of ß-cell mass and function with upregulation of Fas expression in c-Kit(Wv/+) mouse islets, suggesting that there is a critical balance between c-Kit and Fas activation in ß-cells. In the present study, we investigated the interrelationship between c-Kit and Fas activation that mediates ß-cell survival and function. We generated double mutant, c-Kit(Wv/+);Fas(lpr/lpr) (Wv(-/-)), mice to study the physiological and functional role of Fas with respect to ß-cell function in c-Kit(Wv/+) mice. Isolated islets from these mice and the INS-1 cell line were used. We observed that islets in c-Kit(Wv/+) mice showed a significant increase in ß-cell apoptosis along with upregulated p53 and Fas expression. These results were verified in vitro in INS-1 cells treated with SCF or c-Kit siRNA combined with a p53 inhibitor and Fas siRNA. In vivo, Wv(-/-) mice displayed improved ß-cell function, with significantly enhanced insulin secretion and increased ß-cell mass and proliferation compared with Wv(+/+) mice. This improvement was associated with downregulation of the Fas-mediated caspase-dependent apoptotic pathway and upregulation of the cFlip/NF-κB pathway. These findings demonstrate that a balance between the c-Kit and Fas signaling pathways is critical in the regulation of ß-cell survival and function.
Asunto(s)
Apoptosis , Diabetes Mellitus Tipo 1/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Transducción de Señal , Receptor fas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular , Cruzamientos Genéticos , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/fisiopatología , Regulación de la Expresión Génica , Heterocigoto , Secreción de Insulina , Células Secretoras de Insulina/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-kit/genética , Interferencia de ARN , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de Células Madre/antagonistas & inhibidores , Factor de Células Madre/genética , Factor de Células Madre/metabolismo , Técnicas de Cultivo de Tejidos , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Receptor fas/antagonistas & inhibidores , Receptor fas/genéticaRESUMEN
Stem cell factor (SCF) activates hematopoietic stem cell (HSC) self-renewal and is being used to stimulate the ex vivo expansion of HSCs. The mechanism by which SCF supports expansion of HSCs remains poorly understood. In cord blood ex vivo expansion assays, a newly produced anti-SCF monoclonal antibody (clone 23C8) was found to significantly inhibit the expansion of CD34+ cells. This antibody appears to bind directly to a part of SCF that is critical for biological activity toward expansion of CD34+ cells, which is located in the first 104 amino acids from the NH2-terminus.
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Anticuerpos Monoclonales/farmacología , Sangre Fetal/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Proteínas Recombinantes/antagonistas & inhibidores , Factor de Células Madre/antagonistas & inhibidores , Antígenos CD34/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Sangre Fetal/citología , Sangre Fetal/inmunología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Factor de Células Madre/inmunología , Factor de Células Madre/metabolismoRESUMEN
PURPOSE: To study the roles played by stem cell factor (SCF) and SCF receptor c-kit in wound healing of corneal epithelial cells. METHODS: A 2 mm corneal epithelial wound was made in control (WBB6F1(+/+)), SCF (Sl/Sl(d))-, and c-kit (W/W(v)) mutant mice, and the speed of wound healing, 5-bromo-2'-deoxyuridine (BrdU) incorporation, and scanning electron microscopic (SEM) morphology of the corneas were examined. The incorporation of BrdU and the degree of cell attachment in cultured mouse corneal epithelial cells (MCECs) isolated from WBB6F1(+/+), Sl/Sl(d), and W/W(v) mice were examined. Cultured immortalized human corneal epithelial cells (HCECs) were examined by a cell attachment assay after their exposure to anti-SCF antibodies, tyrosine kinase inhibitor (genistein), and competitive Arg-Gly-Asp (RGD) peptide, as well as on cultures treated with extracellular matrix. RESULTS: The speed of corneal wound healing was slower in Sl/Sl(d) and W/W(v) mice than in controls (p<0.01) and the speed of healing in Sl/Sl(d) mice recovered after topical application of SCF (8 ng/ml). No significant difference was found in the BrdU incorporation assay either in vivo or in vitro. Loosened epithelial cells were detected at wound margins in W/W(v) mice by SEM. The cell attachment rate was increased by 157% in cells from WBB6F1(+/+) and 252% in Sl/Sl(d) MCECs by recombinant mouse SCF; however, no significant difference was found in W/W(v) MCECs. Anti-SCF antibodies (Ab), genistein, and RGD peptide reduced the percentage of attached HCECs. Anti-SCF Ab inhibited the attachment of HCECs on fibronectin, laminin, or type IV collagen coated dishes. CONCLUSIONS: These findings indicate that the SCF/c-kit system may play a role in corneal wound healing through epithelial cell attachment.
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Células Epiteliales/efectos de los fármacos , Epitelio Corneal/efectos de los fármacos , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Células Madre/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Anticuerpos/farmacología , Bromodesoxiuridina , Adhesión Celular/efectos de los fármacos , Colágeno Tipo IV/química , Células Epiteliales/metabolismo , Células Epiteliales/patología , Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Fibronectinas/química , Genisteína/farmacología , Laminina/química , Ratones , Ratones Transgénicos , Microscopía Electrónica de Rastreo , Oligopéptidos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Factor de Células Madre/antagonistas & inhibidores , Factor de Células Madre/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
We established a model for the stem cell factor (SCF)-associated stimulation of human epidermal equivalent (HEE) pigmentation. The addition of SCF (at 5 nM) gradually stimulated the visible pigmentation of HEEs over 14 days of treatment. A time course study using real-time RT-PCR and western blotting analysis demonstrated that the expression of all melanocyte-specific genes and proteins examined was gradually up-regulated over 7-10 days of treatment with SCF. The addition of astaxanthin (Ax) at concentrations of 1, 4, or 8 µM markedly abolished the SCF- but not the endothelin (EDN)1-elicited increase in visible pigmentation over 14 days in a dose-dependent manner, with almost complete inhibition at 8 µM. While no degeneration of the epidermal tissue was visible at day 14 by HE staining, melanin deposition throughout the epidermis was markedly reduced in the Ax-treated HEEs at day 14 compared to untreated controls. Ax significantly reduced the eumelanin content of HEEs to the non-SCF-stimulated level at concentrations of 4 or 8 µM compared with untreated controls. Real-time RT-PCR and western blotting of Ax-treated HEEs revealed that the SCF-stimulated expression of tyrosinase (TYR), TYR-related protein-1 (TYRP1), and Pmel17, as well as microphthalmia-associated transcription factor (MITF), is significantly suppressed by Ax at the transcriptional and translational levels. Studies using cultured normal human melanocytes revealed that pre-treatment with Ax interrupts the SCF- but not the EDN1-induced stimulation of TYR activity, and there was no direct inhibitory effect of Ax on TYR activity in vitro. These findings indicate that Ax attenuates SCF-stimulated pigmentation by directly interrupting SCF-associated intracellular signaling linkages through increased expression of MITF, which leads to the stimulated expression of melanogenic genes and proteins in a reactive oxygen species depletion-independent mechanism.
Asunto(s)
Epidermis/efectos de los fármacos , Hiperpigmentación/metabolismo , Melanocitos/metabolismo , Pigmentación de la Piel/efectos de los fármacos , Factor de Células Madre/antagonistas & inhibidores , Células Cultivadas , Endotelina-1/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Melanocitos/efectos de los fármacos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Factor de Células Madre/farmacología , Xantófilas/farmacología , Antígeno gp100 del Melanoma/metabolismoRESUMEN
Estrogen inhibits cyst breakdown and primordial follicle assembling of germ cells, but little is known about the underlying mechanisms. We aimed to analyze the effects of estrogen on the early development of mouse follicles using an in vitro ovary culture system and in vivo injection. Newborn mouse ovarian tissues were cultured in vitro for 2 or 4 days with estrogen of 0 M, 10(-8) M and 10(-4) M, respectively, and neonatal mice were injected with 5mg/kg/day estrogen. We found that the percentages of different-stage follicles significantly varied between the control and estrogen-treated groups. In vitro experiments showed that the unassembled follicles accounted for 70.5±2.7% and the primordial follicles accounted for 29.5±2.7% in the treatment group, but in the control group, ovaries had 61.7±8.4% unassembled follicles. In vivo experiments showed that the percentages of unassembled follicles and primordial follicles were 37.1±5.2% and 51.6±2.4% in the control group, while they were 72.6±5.2% and 25.1±5.5% in the treatment group. Moreover, we analyzed the expression of Kit ligand in mouse ovaries treated by estrogen with real-time PCR and western blot technology, and found that compared with the control group, both mRNA and protein expression levels were decreased in the treatment group (P<0.05). These results indicate that estrogen inhibits the development of mouse ovarian follicles by regulating the expression of Kit ligand.
Asunto(s)
Estrógenos/farmacología , Folículo Ovárico/efectos de los fármacos , Factor de Células Madre/antagonistas & inhibidores , Animales , Femenino , Ratones , Técnicas de Cultivo de Órganos , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factor de Células Madre/biosíntesis , Factor de Células Madre/genéticaRESUMEN
PURPOSE: Radiotherapy resistance is a commonly encountered problem in cancer treatment. In this regard, stabilization of endothelial cells and release of angiogenic factors by cancer cells contribute to this problem. In this study, we used human lung adenocarcinoma (A549) cells to compare the effects of carbon ion and X-ray irradiation on the cells' angiogenic response. METHODS AND MATERIALS: A549 cells were irradiated with biologically equivalent doses for cell survival of either carbon ions (linear energy transfer, 170 keV/µm; energy of 9.8 MeV/u on target) or X-rays and injected with basement membrane matrix into BALB/c nu/nu mice to generate a plug, allowing quantification of angiogenesis by blood vessel enumeration. The expression of angiogenic factors (VEGF, PlGF, SDF-1, and SCF) was assessed at the mRNA and secreted protein levels by using real-time reverse transcription-PCR and enzyme-linked immunosorbent assay. Signal transduction mediated by stem cell factor (SCF) was assessed by phosphorylation of its receptor c-Kit. For inhibition of SCF/c-Kit signaling, a specific SCF/c-Kit inhibitor (ISCK03) was used. RESULTS: Irradiation of A549 cells with X-rays (6 Gy) but not carbon ions (2 Gy) resulted in a significant increase in blood vessel density (control, 20.71 ± 1.55; X-ray, 36.44 ± 3.44; carbon ion, 16.33 ± 1.03; number per microscopic field). Concordantly, irradiation with X-rays but not with carbon ions increased the expression of SCF and subsequently caused phosphorylation of c-Kit in endothelial cells. ISCK03 treatment of A549 cells irradiated with X-rays (6 Gy) resulted in a significant decrease in blood vessel density (X-ray, 36.44 ± 3.44; X-ray and ISCK03, 4.33 ± 0.71; number of microscopic field). These data indicate that irradiation of A549 cells with X-rays but not with carbon ions promotes angiogenesis. CONCLUSIONS: The present study provides evidence that SCF is an X-ray-induced mediator of angiogenesis in A549 cells, a phenomenon that could not be observed with carbon ion irradiation. Thus, in this model system evaluating angiogenesis, carbon ion irradiation may have a therapeutic advantage. This observation should be confirmed in orthotopic lung tumor models.
Asunto(s)
Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/radioterapia , Carbono/uso terapéutico , Radioterapia de Iones Pesados , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/radioterapia , Neovascularización Patológica/etiología , Fotones/uso terapéutico , Adenocarcinoma/metabolismo , Adenocarcinoma del Pulmón , Animales , Carcinoma de Pulmón de Células no Pequeñas/irrigación sanguínea , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Línea Celular Tumoral , Supervivencia Celular/efectos de la radiación , Quimiocina CXCL12/metabolismo , Humanos , Imidazoles/farmacología , Transferencia Lineal de Energía , Neoplasias Pulmonares/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica/metabolismo , Neovascularización Patológica/fisiopatología , Fosforilación , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Células Madre/antagonistas & inhibidores , Factor de Células Madre/metabolismo , Sulfonamidas/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Dysregulated reactive oxygen species (ROS) generation contributes to many human pathologies, including cancer and diabetes. During normal wound repair, inflammation-induced ROS production must be tightly controlled, but the mechanisms reining their generation remain unclear. Herein, we show that transforming growth factor ß-activated kinase 1 (TAK1) directly regulates stem cell factor (SCF) expression, which activates the protein kinase B (PKB)α pro-survival pathway in a cell-autonomous manner to protect keratinocytes from ROS-mediated cell death. TAK1 is a pivotal inflammatory mediator whose expression was transiently elevated during wound healing, paralleling the ROS production profile. TAK1 deficiency in keratinocytes led to increased apoptosis in response to anoikis and TNF-α treatment and was associated with elevated ROS level as analyzed by FACS. Using organotypic skin co-culture and comparative growth factor array analysis, we revealed a cell-autonomous mechanism that involved the SCF/c-Kit/PKBα signaling cascade. Ectopic expression of TAK1 or treatment with exogenous recombinant SCF restored the increased ROS production and apoptotic cell death in TAK1-deficient keratinocytes. Conversely, normal keratinocytes treated with various inhibitors targeting the SCF/c-Kit/PKBα pathway exhibited increased ROS production and TNF-α- or anoikis-induced apoptosis. Our study reveals a novel anti-apoptotic role for SCF in keratinocytes and identifies TAK1 as a novel player uniting inflammation and ROS regulation in skin redox biology.