RESUMEN
Debulking followed by intralesional 5-fluorouracil (deb-IL5FU) is a nonsurgical modality which has been used to treat skin cancer anecdotally for many years. There are few in depth studies examining this technique and success rate of intralesional 5-fluorouracil (IL5FU) for the treatment of cutaneous squamous cell carcinoma (cSCC). To evaluate the response rate of deb-IL5FU for the treatment of cSCC and to determine which patient factors were associated with tumor clearance or treatment failure. A retrospective chart analysis of patients with the diagnosis of cSCC or keratoacanthoma (KA) and subsequent deb-IL5FU treatment. Sixty-one patients with a total of 315 tumors (cSCC and KA), were treated using deb-IL5FU. The overall tumor clearance rate was 89%. This was highest for well-differentiated SCC, SCC, KA-type SCC, and KA. Tumors on the trunk and extremities showed high clearance rates while tumors on the scalp/face/neck/ears showed lower clearance rates. Immunocompetent patients cleared more tumors compared to immunocompromised patients. Limitations included the retrospective nature of this analysis as well as a small sample size. Treatment of cSCC and KA with deb-IL5FU demonstrated high tumor clearance rates. Lower rates of clearance were seen in males, immunosuppressed patients, tumors located on the scalp and face/neck/ears.
Asunto(s)
Carcinoma de Células Escamosas , Queratoacantoma , Neoplasias Cutáneas , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/tratamiento farmacológico , Procedimientos Quirúrgicos de Citorreducción , Fluorouracilo , Humanos , Queratoacantoma/diagnóstico , Queratoacantoma/tratamiento farmacológico , Queratoacantoma/patología , Masculino , Estudios Retrospectivos , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/tratamiento farmacológicoRESUMEN
The pemphigus family of autoimmune bullous disorders is characterized by autoantibody binding to desmoglein 1 and/or 3 (dsg1/dsg3). In this study we show that EGF receptor (EGFR) is activated following pemphigus vulgaris (PV) IgG treatment of primary human keratinocytes and that EGFR activation is downstream of p38 mitogen-activated protein kinase (p38). Inhibition of EGFR blocked PV IgG-triggered dsg3 endocytosis, keratin intermediate filament retraction, and loss of cell-cell adhesion in vitro. Significantly, inhibiting EGFR prevented PV IgG-induced blister formation in the passive transfer mouse model of pemphigus. These data demonstrate cross-talk between dsg3 and EGFR, that this cross-talk is regulated by p38, and that EGFR is a potential therapeutic target for pemphigus. Small-molecule inhibitors and monoclonal antibodies directed against EGFR are currently used to treat several types of solid tumors. This study provides the experimental rationale for investigating the use of EGFR inhibitors in pemphigus.
Asunto(s)
Acantólisis/metabolismo , Receptores ErbB/metabolismo , Regulación de la Expresión Génica , Inmunoglobulina G/metabolismo , Pénfigo/metabolismo , Animales , Animales Recién Nacidos , Adhesión Celular , Células Cultivadas , Desmogleínas/metabolismo , Desmosomas/metabolismo , Detergentes/farmacología , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoglobulina G/química , Queratinocitos/citología , Queratinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Background: Patients with field cancerization will develop numerous superficial non-melanoma skin cancers (NMSCs). Treating patients with field cancerization can be challenging and burdensome due to the numerous non-melanoma skin cancers (NMSCs) they develop and the frequent dermatology visits required for biopsy and treatment. Objective: The success rate of diagnosing and treating lesions suspicious for NMSCs on the same day is measured, immediately after biopsy. Methods: We retrospectively reviewed records of patients with same day lesion diagnosis and curettage treatment to determine diagnostic accuracy, treatment failure, and number needed to treat to reduce a follow-up treatment. Results: A total of 237 lesions underwent same day biopsy and treatment, of which the majority were NMSC (66%) or actinic keratosis (23%). Patients had at least 3 months and a median of 17 months follow-up. A total of 20 lesions either recurred or were deemed to require additional treatment. The number needed to treat (NNT) to prevent one follow-up treatment was 1.3. Limitations: sample size limited ability to determine risk factors for treatment failure. Conclusion: Simultaneous diagnosis and treatment of superficial NMSCs is a successful way of improving efficiency and patient satisfaction.
Asunto(s)
Enfermedad de Bowen/cirugía , Efecto Espectador/inmunología , Cirugía de Mohs , Neoplasias Cutáneas/cirugía , Enfermedad de Bowen/inmunología , Enfermedad de Bowen/terapia , Terapia Combinada , Dedos , Humanos , Huésped Inmunocomprometido , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Masculino , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/terapia , Adulto JovenRESUMEN
Pemphigus vulgaris (PV) is an autoimmune blistering disease in which antibodies against the desmosomal cadherin, DSG3 (desmoglein-3), cause acantholysis. It has become increasingly clear that loss of cell-cell adhesion in PV is a complex and active process involving multiple signaling events such as activation of p38MAPK. It has also been demonstrated that incubating keratinocytes with PV IgG causes a redistribution of DSG3 from the cell surface to endosomes, which target these proteins for degradation. This study was undertaken to determine the relationship between p38MAPK and DSG3 endocytosis in pemphigus. In this work, we confirm that PV IgG causes internalization of cell-surface DSG3 into endosomes (as early as 4 h), which are then depleted from both detergent-soluble and detergent-insoluble pools. Cell-surface DSG3 internalization and depletion from both the detergent-soluble and detergent-insoluble fractions were blocked by the p38MAPK inhibitor SB202190. These data suggest that p38MAPK is capable of regulating PV IgG-mediated DSG3 internalization and that previously isolated mechanistic observations may be linked to a common pathway by which pemphigus autoantibodies lead to acantholysis.
Asunto(s)
Desmogleína 3/metabolismo , Regulación Enzimológica de la Expresión Génica , Pénfigo/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Autoanticuerpos/química , Biotinilación , Membrana Celular/metabolismo , Detergentes/farmacología , Endocitosis , Endosomas/metabolismo , Humanos , Inmunoglobulina G/química , Queratinocitos/citología , Microscopía Confocal/métodos , Transducción de SeñalRESUMEN
Mast cells secrete various substances that initiate and perpetuate allergic responses. Cross-linking of the high-affinity receptor for IgE (FcepsilonRI) in RBL-2H3 and bone marrow-derived mast cells activates sphingosine kinase (SphK), which leads to generation and secretion of the potent sphingolipid mediator, sphingosine-1-phosphate (S1P). In turn, S1P activates its receptors S1P1 and S1P2 that are present in mast cells. Moreover, inhibition of SphK blocks FcepsilonRI-mediated internalization of these receptors and markedly reduces degranulation and chemotaxis. Although transactivation of S1P1 and Gi signaling are important for cytoskeletal rearrangements and migration of mast cells toward antigen, they are dispensable for FcepsilonRI-triggered degranulation. However, S1P2, whose expression is up-regulated by FcepsilonRI cross-linking, was required for degranulation and inhibited migration toward antigen. Together, our results suggest that activation of SphKs and consequently S1PRs by FcepsilonRI triggering plays a crucial role in mast cell functions and might be involved in the movement of mast cells to sites of inflammation.
Asunto(s)
Mastocitos/inmunología , Receptores Acoplados a Proteínas G/genética , Receptores de IgE/metabolismo , Animales , Secuencia de Bases , Degranulación de la Célula , Línea Celular , Quimiotaxis , Lisofosfolípidos/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Interferente Pequeño/genética , Receptores Lisofosfolípidos , Esfingosina/análogos & derivados , Esfingosina/biosíntesis , Activación TranscripcionalRESUMEN
Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, is the ligand for five specific G protein-coupled receptors, named S1P(1) to S1P(5). In this study, we found that cross-communication between platelet-derived growth factor receptor and S1P(2) serves as a negative damper of PDGF functions. Deletion of the S1P(2) receptor dramatically increased migration of mouse embryonic fibroblasts toward S1P, serum, and PDGF but not fibronectin. This enhanced migration was dependent on expression of S1P(1) and sphingosine kinase 1 (SphK1), the enzyme that produces S1P, as revealed by downregulation of their expression with antisense RNA and small interfering RNA, respectively. Although S1P(2) deletion had no significant effect on tyrosine phosphorylation of the PDGF receptors or activation of extracellular signal-regulated kinase 1/2 or Akt induced by PDGF, it reduced sustained PDGF-dependent p38 phosphorylation and markedly enhanced Rac activation. Surprisingly, S1P(2)-null cells not only exhibited enhanced proliferation but also markedly increased SphK1 expression and activity. Conversely, reintroduction of S1P(2) reduced DNA synthesis and expression of SphK1. Thus, S1P(2) serves as a negative regulator of PDGF-induced migration and proliferation as well as SphK1 expression. Our results suggest that a complex interplay between PDGFR and S1P receptors determines their functions.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Receptores de Lisoesfingolípidos/metabolismo , Animales , Estructuras de la Membrana Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/metabolismo , Eliminación de Gen , Expresión Génica , Masculino , Ratones , Modelos Biológicos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptores de Lisoesfingolípidos/deficiencia , Receptores de Lisoesfingolípidos/genética , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Renal transplant recipients are at an increased risk of developing verrucae due to chronic immunosuppression, and certain therapies may confer a greater risk. Herein, we describe a 51-year-old woman with a 10-year-old unrelated kidney transplant who developed numerous therapy-resistant verrucae while on mycophenolate mofetil and tacrolimus maintenance immunosuppression. Over several years of immunosuppressant therapy, she declined the approach of reducing her mycophenolate mofetil dose to potentially improve her verrucae. Unfortunately, she later developed graft rejection requiring reversion to peritoneal dialysis. Within months of reducing her mycophenolate mofetil dose (her tacrolimus dose remained unchanged), she experienced dramatic resolution of many of her verrucae. In the current case, the observed clinical improvement may have resulted from either the total reduction of immunosuppression or the specific reduction of mycophenolate mofetil. Consequently, mycophenolate mofetil may contribute to the refractory nature of verrucae within renal transplant recipients, and further research should determine the relationship between verrucae development and both specific immunosuppressant therapies and the degree of immunosuppression.
Asunto(s)
Inmunosupresores/efectos adversos , Trasplante de Riñón/efectos adversos , Ácido Micofenólico/efectos adversos , Complicaciones Posoperatorias/inducido químicamente , Verrugas/inducido químicamente , Femenino , Rechazo de Injerto/inducido químicamente , Humanos , Terapia de Inmunosupresión/efectos adversos , Terapia de Inmunosupresión/métodos , Inmunosupresores/administración & dosificación , Persona de Mediana Edad , Ácido Micofenólico/administración & dosificación , Tacrolimus/administración & dosificación , Tacrolimus/efectos adversosRESUMEN
While most of the pharmacological therapies for melanoma utilize the apoptotic machinery of the cells, the available therapeutic options are limited due to the ability of melanoma cells to resist programmed cell death. Human melanoma cell lines A-375 and M186 are sensitive to ceramide- and Fas-induced cell death, while Mel-2a and M221 are resistant. We have now found that Mel-2a and M221 cells have a significantly higher ceramide/sphingosine-1-phosphate (S1P) ratio than A-375 and M186 cells. As sphingosine kinase (SphK) type 1 plays a critical role in determining the dynamic balance between the proapoptotic sphingolipid metabolite ceramide and the prosurvival S1P, we examined its role in apoptosis of melanoma cells. Increasing SphK1 expression reduced the sensitivity of A-375 melanoma cells to Fas- and ceramide-mediated apoptosis. Conversely, downregulation of SphK1 with small interfering RNA decreased the resistance of Mel-2a cells to apoptosis. Importantly, overexpression of the prosurvival protein Bcl-2 in A-375 cells markedly stimulated SphK1 expression and activity, while downregulation of Bcl-2 reduced SphK1 expression. This link between Bcl-2 and SphK1 might be an additional clue to chemotherapy resistance of malignant melanoma.
Asunto(s)
Apoptosis/fisiología , Ceramidas/metabolismo , Melanoma/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Regulación hacia Abajo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Células Tumorales CultivadasRESUMEN
Asthma is a complex condition in which exposure to environmental antigens induces inflammatory reactions in the airway characterized by activation of mast cells and eosinophils. Mast cells are known to be the main effector cells in eliciting IgE-mediated allergic response. These cells secrete various substances that perpetuate inflammation and provoke airway smooth muscle (ASM) contraction. A newly recognized addition to the repertoire of FcepsilonRI-mediated signaling events is the activation of sphingosine kinase leading to the generation of the potent sphingolipid mediator, sphingosine-1-phosphate (S1P) from sphingosine. S1P secretion by the lung significantly increases after challenge with an allergen, adding this sphingolipid metabolite to the variety of mediators that are released during an allergic reaction [FASEB J. 15 (2001) 1212]. Indeed, similar to previous reports, we found that FcepsilonRI cross-linking not only increased cellular levels of S1P, it also markedly enhanced its secretion from rat basophilic leukemia RBL-2H3 cells. Moreover, S1P induced degranulation of RBL and bone marrow derived mast cells (BMMCs) cells as determined by hexosaminidase release. Treatment of BMMCs with the sphingosine kinase inhibitors, DL-threo-dihydrosphingosine and dimethylsphingosine, reduced IgE/Ag stimulated histamine release. RT-PCR analysis demonstrated that these mast cells express S1P receptors EDG-1 and EDG-5 but not EDG-3, EDG-6 or EDG-8 transcripts. Further studies are needed to determine whether IgE triggering results in transactivation of EDG-1 or EDG-5 present on mast cells and whether this is a critical event for mast cell activation.
Asunto(s)
Asma/inmunología , Lisofosfolípidos , Mastocitos/inmunología , Esfingosina/análogos & derivados , Esfingosina/fisiología , Obstrucción de las Vías Aéreas/inmunología , Animales , Asma/fisiopatología , Inflamación/inmunología , Pulmón/citología , Pulmón/fisiopatología , Modelos Inmunológicos , Músculo Liso/fisiopatología , Ratas , Receptores de IgE/metabolismo , Transducción de Señal , Esfingolípidos/metabolismoRESUMEN
BACKGROUND: Vismodegib is a novel hedgehog pathway inhibitor approved to treat advanced and metastatic basal cell carcinoma (BCC) in the United States. Several studies have demonstrated efficacy for treatment of new and existing BCC in both basal cell nevus syndrome (BCNS) and non-BCNS patients. However, severe and numerous adverse events are associated with vismodegib use. Therefore, we have also examined all of the currently published clinical trials and tabulated the available adverse events for review. The most frequently reported adverse events include muscle spasms (53.4%), dysgeusia/ageusia (49.3%), alopecia (38.8%), fatigue (32.0%), nausea (28.4%), weight loss (24.2%), and decreased appetite (16.5%). CASE STUDY: We report a case of a previously healthy 72-year-old male with a history of innumerable BCCs who developed severe nausea, jaundice, and cholestasis with significantly elevated BUN, creatinine, and liver enzymes one month after starting vismodegib. The patient began using over-the-counter nonsteroidal anti-inflammatory drugs (NSAIDs) to treat severe, vismodegib-induced myalgia. No other new medications were started. Our patient had no history of liver disease. CONCLUSIONS: Herein, we describe a potential serious adverse effect associated with vismodegib use. Whether the illness is directly attributable to the medication or the result of drug-drug interactions between vismodegib and NSAIDs, practitioners should be aware of the possibility of hepatic injury in patients on vismodegib. Furthermore, patients need to be informed of the potential risks of vismodegib and should be monitored closely to ensure that life-threatening complications of treatment are avoided.
Asunto(s)
Anilidas/efectos adversos , Antiinflamatorios no Esteroideos/efectos adversos , Antineoplásicos/efectos adversos , Carcinoma Basocelular/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Colestasis/inducido químicamente , Piridinas/efectos adversos , Neoplasias Cutáneas/tratamiento farmacológico , Anciano , Aspirina/efectos adversos , Interacciones Farmacológicas , Disgeusia/inducido químicamente , Humanos , Masculino , Mialgia/inducido químicamente , Mialgia/tratamiento farmacológico , Naproxeno/efectos adversosRESUMEN
In pemphigus vulgaris and pemphigus foliaceus (PF), autoantibodies against desmoglein-3 and desmoglein-1 induce epidermal cell detachment (acantholysis) and blistering. Activation of keratinocyte intracellular signaling pathways is emerging as an important component of pemphigus IgG-mediated acantholysis. We previously reported activation of p38 mitogen-activated protein kinase (MAPK) in response to pathogenic pemphigus vulgaris and PF IgG. Inhibition of p38MAPK blocked pemphigus IgG-induced cytoskeletal reorganization in tissue culture and blistering in pemphigus mouse models. We now extend these observations by demonstrating two peaks of p38MAPK activation in pemphigus tissue culture and mouse models. Administration of the p38MAPK inhibitor SB202190 before PF IgG injection blocked both peaks of p38MAPK phosphorylation and blister formation, consistent with our previous findings; however, administration of the inhibitor 4 h after PF IgG injection blocked only the later peak of p38MAPK activation but failed to block blistering. Examination of the temporal relationship of p38MAPK phosphorylation and apoptosis showed that apoptosis occurs at or after the second peak of p38MAPK activation. The time course of p38MAPK activation and apoptotic markers, as well as the ability of inhibitors of p38MAPK to block activation of the proapoptotic proteinase caspase-3, suggest that activation of apoptosis is downstream to, and a consequence of, p38MAPK activation in pemphigus acantholysis. Furthermore, these observations suggest that the earlier peak of p38MAPK activation is part of the mechanism leading to acantholysis, whereas the later peak of p38MAPK and apoptosis may not be essential for acantholysis.
Asunto(s)
Acantólisis/enzimología , Pénfigo/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Acantólisis/patología , Animales , Apoptosis/efectos de los fármacos , Autoanticuerpos/metabolismo , Caspasa 3/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/patología , Desmogleína 1/metabolismo , Desmogleína 3/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Humanos , Inmunoglobulina G/metabolismo , Ratones , Pénfigo/patología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Técnicas de Cultivo de Tejidos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Mast cells play a central role in inflammatory and immediate-type allergic reactions by secreting a variety of biologically active substances, including sphingosine-1 phosphate (S1P). Sphingosine kinase 1 (SphK1) and formation of S1P, which leads to transactivation of S1P receptors and their downstream signaling pathways, regulates mast-cell functions initiated by cross-linking of the high-affinity immunoglobulin E (IgE) receptor FcepsilonRI. Surprisingly, overexpression of SphK1 in rat basophilic leukemia (RBL)-2H3 mast cells impaired degranulation as well as migration toward antigen. These effects were reversed by serum withdrawal, yet the increased formation and secretion of S1P were the same as in the presence of serum. Nonetheless, serum increased localization of SphK1 at the plasma membrane. This restricted formation of S1P induced internalization and desensitization of S1P receptors on the surface of mast cells as determined by confocal immunofluorescence microscopy, aberrant S1P receptor signaling, and lack of S1P receptor coupling to G proteins. Serum starvation, which significantly reduced membrane-associated SphK1 activity, restored S1P receptor functions. Our results have important implications for mast-cell migration and degranulation as well as for the biologic functions of the S1P receptors on cells that are circulating in the bloodstream.
Asunto(s)
Mastocitos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Receptores de Lisoesfingolípidos/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Movimiento Celular , Quimiotaxis , Reactivos de Enlaces Cruzados/farmacología , Medio de Cultivo Libre de Suero/farmacología , Regulación hacia Abajo , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Inmunoglobulina E/química , Inflamación , Ligandos , Microscopía Confocal , Microscopía Fluorescente , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fenotipo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transporte de Proteínas , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Receptores de IgE/química , Transducción de Señal , Factores de Tiempo , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
We had found previously that neurotrophin-3 (NT-3) is a potent stimulator of cAMP-response element binding protein (CREB) phosphorylation in cultured oligodendrocyte progenitors. Here, we show that CREB phosphorylation in these cells is also highly stimulated by sphingosine-1-phosphate (S1P), a sphingolipid metabolite that is known to be a potent mediator of numerous biological processes. Moreover, CREB phosphorylation in response to NT-3 involves sphingosine kinase 1 (SphK1), the enzyme that synthesizes S1P. Immunocytochemistry and confocal microscopy indicated that NT-3 induces translocation of SphK1 from the cytoplasm to the plasma membrane of oligodendrocytes, a process accompanied by increased SphK1 activity in the membrane fraction where its substrate sphingosine resides. To examine the involvement of SphK1 in NT-3 function, SphK1 expression was down-regulated by treatment with SphK1 sequence-specific small interfering RNA. Remarkably, the capacity of NT-3 to protect oligodendrocyte progenitors from apoptotic cell death induced by growth factor deprivation was abolished by down-regulating the expression of SphK1, as assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Altogether, these results suggest that SphK1 plays a crucial role in the stimulation of oligodendrocyte progenitor survival by NT-3, and demonstrate a functional link between NT-3 and S1P signaling, adding to the complexity of mechanisms that modulate neurotrophin function and oligodendrocyte development.
Asunto(s)
Neurotrofina 3/farmacología , Oligodendroglía , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Células Madre/efectos de los fármacos , Animales , Animales Recién Nacidos , Western Blotting/métodos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Interacciones Farmacológicas , Regulación de la Expresión Génica/efectos de los fármacos , Etiquetado Corte-Fin in Situ/métodos , Lisofosfolípidos/farmacología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Fosforilación/efectos de los fármacos , Proteína Quinasa C/farmacología , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Esfingosina/análogos & derivados , Esfingosina/farmacología , Células Madre/fisiología , Tiazoles/farmacología , Tiazolidinedionas , Tionas/farmacología , Factores de TiempoRESUMEN
Human ceramide kinase was recently cloned and characterized. Recombinant ceramide kinase is highly active and ceramide is the only lipid that it phosphorylates, indicating that it should be useful for the measurement of ceramide levels in biological samples by conversion to ceramide-1-phosphate, in a manner analogous to that of the widely used Escherichia coli diacylglycerol kinase method. Using recombinant ceramide kinase, we have now developed a rapid and specific enzymatic method to quantify mass levels of long-chain ceramides in cellular lipid extracts. This new ceramide kinase assay is more specific than the commonly used diacylglycerol kinase method because the ubiquitous lipid diacylglycerol, the preferred substrate for diacyglycerol kinase which is usually present at higher concentrations than ceramide and can interfere with ceramide phosphorylation, is completely inactive with ceramide kinase. Moreover, this high specificity eliminates the need for analysis of the lipid product by thin-layer chromatography since ceramide-1-phosphate is the only radiolabeled lipid in organic solvent extracts of ceramide kinase reactions.
Asunto(s)
Bioensayo , Ceramidas/análisis , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ceramidas/metabolismo , Humanos , Radioisótopos de Fósforo/metabolismoRESUMEN
Sphingosine-1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors that regulate a wide variety of cellular functions, including cytoskeletal rearrangements and cell motility. Because of the pivotal role of S1P, its levels are low and tightly regulated in a spatial-temporal manner through its synthesis catalyzed by sphingosine kinases and degradation by an S1P lyase and specific S1P phosphatases (SPP). Surprisingly, down-regulation of SPP-1 enhanced migration toward epidermal growth factor (EGF); conversely, overexpression of SPP-1, which is localized in the endoplasmic reticulum, attenuated migration toward EGF. To determine whether the inhibitory effect on EGF-induced migration was because of decreased S1P or increased ceramide as a consequence of acylation of increased sphingosine by ceramide synthase, we used fumonisin B1, a specific inhibitor of ceramide synthase. Although fumonisin B1 blocked ceramide production and increased sphingosine, it did not reverse the negative effect of SPP-1 expression on EGF- or S1P-induced chemotaxis. EGF activated the epidermal growth factor receptor to the same extent in SPP-1-expressing cells, yet ERK1/2 activation was impaired. In agreement, PD98059, an inhibitor of the ERK-activating enzyme MEK, decreased EGF-stimulated migration. We next examined the possibility that intracellularly generated S1P might be involved in activating a G protein-coupled S1P receptor important for EGF-directed migration. Treatment with pertussis toxin to inactivate Galpha(i) suppressed EGF-induced migration. Moreover, expression of regulator of G protein signaling 3, which inhibits S1P receptor signaling and completely prevented ERK1/2 activation mediated by S1P receptors, not only reduced migration toward S1P but also markedly reduced migration toward EGF. Collectively, these results suggest that metabolism of S1P by SPP-1 is important for EGF-directed cell migration.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Proteínas de la Membrana/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Western Blotting , Línea Celular , Movimiento Celular , Ceramidas/metabolismo , Quimiotaxis , Cromatografía en Capa Delgada , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Receptores ErbB/metabolismo , Flavonoides/farmacología , Fumonisinas/farmacología , Vectores Genéticos , Humanos , Hidrólisis , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oxidorreductasas/metabolismo , Toxina del Pertussis/farmacología , Pruebas de Precipitina , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
The lysophospholipids, lysophosphatidic acid, sphingosine-1-phosphate, and sphingosylphosphorylcholine (SPC), are bioactive lipid molecules that regulate diverse biological processes. Although the specific G protein-coupled receptors for lysophosphatidic acid and sphingosine-1-phosphate have been well-characterized, much less is known of the SPC receptors. It has been reported that ovarian cancer G protein-coupled receptor 1 (OGR1) is a high affinity receptor for SPC, and its closely related homologue GPR4 is a high affinity receptor for SPC with low affinity for lysophosphatidylcholine (LPC). However, in a functional assay to examine the specificity of ligand binding, we found that neither SPC nor LPC, or other related lysophospholipids, induced internalization of GPR4 from the plasma membrane. In agreement, these lysolipids also did not induce translocation of beta-arrestin2-GFP from the cytosol to the plasma membrane in GPR4 expressing cells. However, when these cells were cotransfected with G protein-coupled receptor kinase 2, in the absence of added ligands, beta-arrestin2-GFP accumulated in cytoplasmic vesicles, reminiscent of vesicular labeling usually observed after agonist stimulation of GPCRs. In addition, neither SPC nor LPC stimulated the binding of GTPgammaS to membranes prepared from GPR4 expressing cells and did not activate ERK1/2. Surprisingly, enforced expression of GPR4 inhibited activation of ERK1/2 induced by several stimuli, including SPC, sphingosine-1-phosphate, and even EGF. Collectively, our results suggest that SPC and LPC are not the ligands for GPR4 and that this receptor may constitutively inhibit ERK1/2 activation.