RESUMEN
The main targets of hepatitis C virus (HCV) are hepatocytes, the highly polarized cells of the liver, and all the steps of its life cycle are tightly dependent on host lipid metabolism. The interplay between polarity and lipid metabolism in HCV infection has been poorly investigated. Signaling lipids, such as phosphoinositides (PIs), play a vital role in polarity, which depends on the distribution and expression of PI kinases and PI phosphatases. In this study, we report that HCV core protein, expressed in Huh7 and Madin-Darby canine kidney (MDCK) cells, disrupts apicobasal polarity. This is associated with decreased expression of the polarity protein Dlg1 and the PI phosphatase SHIP2, which converts phosphatidylinositol 3,4,5-trisphosphate into phosphatidylinositol 4,5-bisphosphate (PtdIns(3,4)P2). SHIP2 is mainly localized at the basolateral membrane of polarized MDCK cells. In addition, PtdIns(3,4)P2 is able to bind to Dlg1. SHIP2 small interfering RNA or its catalytically dead mutant disrupts apicobasal polarity, similar to HCV core. In core-expressing cells, RhoA activity is inhibited, whereas Rac1 is activated. Of interest, SHIP2 expression rescues polarity, RhoA activation, and restricted core level in MDCK cells. We conclude that SHIP2 is an important regulator of polarity, which is subverted by HCV in epithelial cells. It is suggested that SHIP2 could be a promising target for anti-HCV treatment.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Epiteliales/metabolismo , Hepacivirus/fisiología , Hepatocitos/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas del Núcleo Viral/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular , Polaridad Celular , Homólogo 1 de la Proteína Discs Large , Perros , Células Epiteliales/virología , Regulación de la Expresión Génica , Hepatocitos/virología , Interacciones Huésped-Patógeno , Humanos , Proteínas de la Membrana/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/genética , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteínas del Núcleo Viral/genéticaRESUMEN
Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of phosphatidic acid (PA), which is itself a source of diacylglycerol (DAG). These two versatile lipid second messengers are at the centre of a phospholipid signalling network and as such are involved in several cellular functions. However, their role in T-cell activation and functions are still enigmatic. In order to elucidate this role, we generated a human and a murine T-cell line that stably overexpressed the PLD2 isoform. Analysis of the Ras-MAPK pathway upon phorbol myristate acetate (PMA) and ionomycin stimulation revealed that PLD2 promoted an early and sustained increase in ERK1/2 phosphorylation in both cell lines. This response was inhibited by 1-butanol, a well known distracter of PLD activity, or upon overexpression of a dominant negative PLD2, and it was concomitant with a boost of PA/DAG production. As a functional consequence of this PLD2-dependent MAPK activation, interleukin-2 production evoked by PMA/ionomycin stimulation or CD3/CD28 engagement was enhanced in the two T-cell lines overexpressing PLD2. Thus, PLD2 emerged as an early player upstream of the Ras-MAPK-IL-2 pathway in T-cells via PA and DAG production, raising new possibilities of pharmacological manipulation in immune disorders.
Asunto(s)
Interleucina-2/inmunología , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfolipasa D/metabolismo , Linfocitos T/enzimología , Linfocitos T/inmunología , Animales , Activación Enzimática , Humanos , Ionomicina/metabolismo , Ionóforos/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Células Jurkat , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Fosfolipasa D/genética , Linfocitos T/citología , Acetato de Tetradecanoilforbol/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Lysophosphatidic acid (LPA) is present in abundance in serum resulting from platelet activation and is also found in other biological fluids. LPA controls numerous cellular responses and plays a role in specific functions such as wound healing, especially in the skin. Nevertheless, its presence in the skin has never been investigated. Since re-epithelialization occurs after blister rupture, we tested the presence of endogenous LPA in blister fluid and investigated a possible mechanism for its biosynthesis and biological functions. Using a radioenzymatic assay, LPA was detected in 33 blister fluids originating from 24 bullous dermatoses, and at higher concentrations than in plasma. In parallel, blister fluids contained a lysophospholipase D (LPLD) activity but no detectable phospholipase A2 activity. The expressions of the LPLD autotaxin (ATX) and of LPA1-receptor (LPA1-R) were greatly increased in blister skin when compared with normal skin. Finally, LPA was found to have a positive effect on the migration of cultured keratinocytes. These results show that LPA is present in blister fluid synthesized by the LPLD ATX. Due to its ability to enhance keratinocyte migration, LPA in blister fluid could, via the LPA1-R, play an important role in re-epithelialization occurring after blister rupture.
Asunto(s)
Vesícula/enzimología , Glucosa-6-Fosfato Isomerasa/metabolismo , Glicoproteínas/metabolismo , Lisofosfolípidos/biosíntesis , Complejos Multienzimáticos/metabolismo , Piel/enzimología , Adulto , Anciano , Anciano de 80 o más Años , Vesícula/genética , Vesícula/metabolismo , Movimiento Celular , Células Cultivadas , Femenino , Glucosa-6-Fosfato Isomerasa/genética , Glicoproteínas/genética , Humanos , Queratinocitos/efectos de los fármacos , Lisofosfolípidos/análisis , Lisofosfolípidos/farmacología , Masculino , Persona de Mediana Edad , Complejos Multienzimáticos/genética , Fosfodiesterasa I , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Piel/química , Piel/metabolismo , Cicatrización de HeridasRESUMEN
The lipid mediator sphingosine 1-phosphate (S1P) may alter the proliferation of mesangial cells during pathophysiological processes. Here, S1P stimulated proliferation of rat mesangial cells and phosphorylation of MAPKs at subconfluent cell density. Both effects were inhibited by pertussis toxin treatment. Mesangial cells expressed several S1P receptors of the endothelial differentiation gene family: EDG-1, -3, -5, and -8. Conversely, S1P induced apoptosis at low cell density (2 x 10(4) cells/cm(2)), which was demonstrated by flow cytometry and Hoechst staining. Apoptosis was observed also in quiescent or growing cells and was not reverted by lysophosphatidic acid or platelet-derived growth factor. S1P enhanced phosphorylation of SAPKs. Incubation with [(33)P]S1P, [(3)H]S1P, and [(3)H]sphingosine demonstrated increased S1P hydrolysis, resulting in enhanced intracellular sphingosine levels and decreased S1P levels. A rise in total ceramide levels was also observed; however, ceramide did not originate from [(3)H]sphingosine, and S1P-induced apoptosis was not inhibited by fumonisin B, precluding involvement of de novo ceramide synthesis in apoptosis. Therefore, we suggest that sphingosine accumulation and decreased S1P are primarily responsible for S1P-induced apoptosis. In conclusion, incubation of low-density mesangial cells with S1P results in apoptosis, presumably due to increased S1P hydrolysis.