RESUMEN
Dehydroepiandrosterone sulfate (DHEAS) is the most abundant steroid in the human circulation and is secreted by the adrenals in an age-dependent fashion, with maximum levels during the third decade and very low levels in old age. DHEAS is considered an inactive metabolite, whereas cleavage of the sulfate group generates dehydroepiandrosterone (DHEA), a crucial sex steroid precursor. However, here we show that DHEAS, but not DHEA, increases superoxide generation in primed human neutrophils in a dose-dependent fashion, thereby impacting on a key bactericidal mechanism. This effect was not prevented by coincubation with androgen and estrogen receptor antagonists but was reversed by the protein kinase C inhibitor Bisindolylmaleimide 1. Moreover, we found that neutrophils are unique among leukocytes in expressing an organic anion-transporting polypeptide D, able to mediate active DHEAS influx transport whereas they did not express steroid sulfatase that activates DHEAS to DHEA. A specific receptor for DHEAS has not yet been identified, but we show that DHEAS directly activated recombinant protein kinase C-beta (PKC-beta) in a cell-free assay. Enhanced PKC-beta activation by DHEAS resulted in increased phosphorylation of p47(phox), a crucial component of the active reduced nicotinamide adenine dinucleotide phosphate complex responsible for neutrophil superoxide generation. Our results demonstrate that PKC-beta acts as an intracellular receptor for DHEAS in human neutrophils, a signaling mechanism entirely distinct from the role of DHEA as sex steroid precursor and with important implications for immunesenescence, which includes reduced neutrophil superoxide generation in response to pathogens.
Asunto(s)
Sulfato de Deshidroepiandrosterona/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Proteína Quinasa C/metabolismo , Superóxidos/metabolismo , Células Cultivadas , Humanos , Indoles/farmacología , Masculino , Maleimidas/farmacología , NADPH Oxidasas/genética , Neutrófilos/enzimología , Transportadores de Anión Orgánico/genética , Fosforilación/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C betaRESUMEN
Ingenol 3-angelate (PEP005) is a selective small molecule activator of protein kinase C (PKC) extracted from the plant Euphorbia peplus, whose sap has been used as a traditional medicine for the treatment of skin conditions including warts and cancer. We report here that PEP005 also has potent antileukemic effects, inducing apoptosis in myeloid leukemia cell lines and primary acute myeloid leukemia (AML) cells at nanomolar concentrations. Of importance, PEP005 did not induce apoptosis in normal CD34+ cord blood myeloblasts at up to 2-log concentrations higher than those required to induce cell death in primary AML cells. The effects of PEP005 were PKC dependent, and PEP005 efficacy correlated with expression of PKC-delta. The delta isoform of PKC plays a key role in apoptosis and is therefore a rational potential target for antileukemic therapies. Transfection of KG1a leukemia cells, which did not express PKC-delta or respond to PEP005, with enhanced green fluorescent protein (EGFP)-PKC-delta restored sensitivity to induction of apoptosis by PEP005. Our data therefore suggest that activation of PKC-delta provides a novel approach for treatment of acute myeloid leukemia and that screening for PKC-delta expression may identify patients for potential responsiveness to PEP005.
Asunto(s)
Diterpenos/uso terapéutico , Ésteres/uso terapéutico , Leucemia/tratamiento farmacológico , Proteína Quinasa C/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Diterpenos/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Ésteres/farmacología , Humanos , Leucemia/patología , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/patología , Pronóstico , Proteína Quinasa C/genética , Proteína Quinasa C-delta , Transfección , Células Tumorales CultivadasRESUMEN
Protein kinase C (PKC) is a family of 11 isoenzymes that are differentially involved in the regulation of cell proliferation. PKC-betaII, a mitotic lamin kinase, has been shown previously to translocate to the nucleus at G(2)/M and this was coupled to the generation of nuclear diacylglycerol. However, it is not clear how isoenzyme selective translocation and nuclear targeting is achieved during cell cycle. To investigate further the role of nuclear diacylglycerol we measured PKC isoenzyme translocation and analysed diacylglycerol species at different stages of the cell cycle in U937 cells synchronized by centrifugal elutriation. Translocation of PKC-betaII to the membrane fraction, an indicator of activation, occurred at S and G(2)/M, although PKC-betaII was targeted to the nucleus only at G(2)/M. Levels of nuclear diacylglycerol, specifically tetraunsaturated species, increased during G(2)/M. By contrast, there were no obvious changes in nuclear phosphatidic acid species or mass. 1-stearoyl, 2-arachidonyl glycerol (SAG), the major polyunsaturated nuclear diacylglycerol, was able to activate classical PKC isoenzymes (PKC-alpha and beta), but was less effective for activation of novel isoenzymes (PKC-delta), in an in vitro PKC assay. We propose that PKC-betaII nuclear translocation during G(2)/M phase transition is mediated in part by generation of SAG at the nucleus.