RESUMO
Fc gammaRIIA expressed on neutrophils and monocytes has a fundamental role in combating bacterial infections. In the present study, the requirement of cytosolic phospholipase A2 (cPLA2) for induction of Fc gammaRIIA expression was studied in a model of cPLA2-deficient PLB-985 cells (PLB-D cells). Fc gammaRIIA was acquired only during differentiation of PLB but not of PLB-D cells induced by either 1,25-dihydroxyvitamin D3, retinoic acid, or interferon gamma. Addition of prostaglandin E2 (PGE2) to PLB-D cells undergoing differentiation restored the expression of Fc gammaRIIA protein, whereas addition of indomethacin to PLB cells during differentiation inhibited both the production of PGE2 and the expression of Fc gammaRIIA. Inhibition of PKA during PLB differentiation prevented Fc gammaRIIA expression, whereas dibutyryl cAMP (dbcAMP) induced its expression in both PLB and PLB-D cells. CREB phosphorylation and CREB-CRE interaction were detected only in differentiated PLB cells and not PLB-D cells and were inhibited by indomethacin. A reporter gene containing a Fc gammaRIIA gene promoter fragment with the CRE element was sufficient for CREB activation. Our results are the first to show that CREB activation is involved in up-regulation of Fc gammaRIIA expression in myeloid lineages. PGE2 formed via cPLA2 activates CREB through PKA and this process is dependent on development of PGE2 receptor 4.
Assuntos
Antígenos CD/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dinoprostona/farmacologia , Neutrófilos/imunologia , Fosfolipases A/metabolismo , Receptores de IgG/genética , Diferenciação Celular , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/efeitos dos fármacos , Citosol/enzimologia , Primers do DNA , Regulação da Expressão Gênica , Humanos , Cinética , Monócitos/citologia , Monócitos/fisiologia , Neutrófilos/citologia , Fagocitose , Fosfolipases A2 , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The white-rot fungus Pleurotus ostreatus produces two types of extracellular peroxidases: manganese-dependent peroxidase (MnP) and versatile peroxidase (VP). The effect of Mn2+ on fungal growth, peroxidase activity profiles, and lignin degradation by P. ostreatus was studied in liquid culture and under solid-state fermentation conditions on perlite, the latter resembling the natural growth conditions of this fungus. The fungus was grown in either a defined asparagine-containing basidiomycete selective medium (BSM) or in a rich peptone medium (PM). Biomass production, as determined by respiration experiments in solid-state fermentation and liquid cultures and fungal growth on Petri dishes, was higher in the PM than in the BSM. Mn2+ affected biomass production only in the PM on Petri dishes. In the nonamended PM, high levels of MnP and VP activity were detected relative to the nonamended BSM. Nevertheless, a higher rate of 14C-lignin mineralization was measured in the Mn2+-amended BSM, as determined during the course of 47 d of fermentation. Mn2+ amendment of the PM increased mineralization rate to that obtained in the Mn2+-amended BSM. The enzyme activity profiles of MnP and VP were studied in the BSM using anion-exchange chromatography. In the nonamended BSM, only minute levels of MnP and VP were detected. On Mn2+ amendment, two MnP isoenzymes (B1 and B2) appeared. Isoenzyme B2 was purified and showed 100% identity with the MnP isoenzyme purified in our previous study from PM-solid-state fermentation (P6). P6 was found to be the dominant isoenzyme in terms of activity level and gene expression compared with the VP isoenzymes. Based on these results, we concluded that Mn2+ plays a key role in lignin degradation under different nutritional and growth conditions, since it is required for the production of MnP in P. ostreatus.
