BBA - Molecular Cell Research (v.1692, #1)
Editorial Board (ii).
Differential effects on cellular iron metabolism of the physiologically relevant diatomic effector molecules, NO and CO, that bind iron by Ralph N Watts; Des R Richardson (1-15).
Both nitrogen monoxide (NO) and carbon monoxide (CO) are biologically relevant diatomic effector molecules that mediate a variety of biological functions through their avid binding to iron (Fe). Previous studies showed that NO can inhibit Fe uptake from transferrin (Tf) and increase Fe mobilisation from cells [J. Biol. Chem. 276 (2001) 4724]. We used CO gas, a CO-generating agent ([Ru(CO)3Cl2]2), and cells stably transfected with the CO-producing enzyme, haem oxygenase 1 (HO1), to assess the effect of CO on Fe metabolism. These results were compared to the effects of NO produced by a variety of NO-generating agents, including S-nitrosoglutathione (GSNO), spermine-NONOate (SperNO) and S-nitroso-N-acetylpenicillamine (SNAP). Incubation of cells with CO inhibited 59Fe uptake from 59Fe–Tf by cells, and like NO, reduced ATP levels. Hence, the ability of both agents to inhibit 59Fe uptake may be partially mediated by inhibition of energy-dependent processes. These results showing a CO-mediated decrease in 59Fe uptake from 59Fe–Tf using exogenous CO were in agreement with studies implementing cells transfected with HO1. Like NO, CO markedly prevented 59Fe uptake into ferritin. In comparison to the avid ability of exogenous CO to inhibit 59Fe uptake, it had less effect on cellular 59Fe mobilisation. Experiments with HO1-transfected cells compared to control cells showed that 59Fe mobilisation was slightly enhanced. In contrast to NO, CO did not affect the RNA-binding activity of the iron regulatory protein 1 that plays an important role in Fe homeostasis. Our studies demonstrate that subtle differences in the chemistry of NO and CO results in divergence of their ability to affect Fe metabolism.
Keywords: Transferrin; Iron; Iron metabolism; Iron trafficking; Nitric oxide; Carbon monoxide;
Activation of the interleukin-6 promoter by a dominant negative mutant of c-Jun by Laura Faggioli; Chiara Costanzo; Massimo Donadelli; Marta Palmieri (17-24).
The human IL-6 promoter contains multiple regulatory elements such as those binding transcription factors belonging to the NF-κB (−75/−63), C/EBP (−158/−145 and −87/−76) and AP-1 (−283/−277) families. Herein, we report that ectopic expression of c-Jun, C/EBPδ, and the p65 subunit of NF-κB synergistically activates an IL-6 promoter construct containing only a TATA box and a κB binding site. These results suggest that interactions among NF-κB, C/EBP, and AP-1, which are all activated by the most powerful physiological inducers of the IL-6 gene, namely TNF-α and IL-1, may be crucial for maximal activation of the IL-6 promoter in response to the two cytokines. Furthermore, we show that a mutated form of c-Jun lacking the transactivation domain (TAM-67) was a much stronger activator of the IL-6 promoter than c-Jun. In combination with p65 and/or C/EBPδ, TAM-67 also synergistically activated the IL-6 promoter, while it inhibited TNF-α induced AP-1 activity directing an AP-1-responsive reporter plasmid. Lastly, electrophoretic mobility shift assay (EMSA) results strongly suggest the formation of complexes between p65, C/EBPδ, and/or c-Jun or TAM-67 on the κB site, supporting the idea that the functional synergism is determined by a physical interaction. These data provide new insight into the molecular mechanisms regulating the formation of the transcription complex responsible for IL-6 promoter activation.
Keywords: Interleukin-6; Transcription; Transfection; NF-κB; C/EBP; AP-1;
Activation of extracellular-signal regulated kinase is required for phagocytosis by Lymnaea stagnalis haemocytes by Louise D Plows; Richard T Cook; Angela J Davies; Anthony J Walker (25-33).
Haemocytes are the primary defence cells of molluscs. In the present study, extracellular-signal regulated kinase (ERK) 1/2-like proteins were identified within Lymnaea stagnalis haemocytes, with apparent molecular weights of 44 and 43 kDa, respectively. Mitogen-activated protein kinase (MAPK) activity assays have confirmed that the L. stagnalis ERK possesses kinase activity towards Elk-1. Challenge of haemocytes with bacterial lipopolysaccharide (LPS) resulted in a transient activation of ERK, and immunocytochemistry revealed that phospho-ERK was present in both the perinuclear region and the nucleus following challenge. MAPK/ERK kinase (MEK) inhibitors blocked ERK activation confirming that MEK lies upstream of ERK in haemocytes. Moreover, phagocytosis assays, using various inhibitors, showed that ERK activity was vital for efficient phagocytosis and that ERK may be activated by both Ras-dependent and Ras-independent mechanisms. Overall, this study has furthered knowledge of ERK signalling in molluscan immunity and has shown that the ERK pathway regulates the phagocytic activity of molluscan haemocytes.
Keywords: Lymnaea stagnalis; Mollusc; Mitogen-activated protein kinase; Invertebrate; Immunity; Lipopolysaccharide;
Dual role for poly(ADP-ribose)polymerase-1 and -2 and poly(ADP-ribose)glycohydrolase as DNA-repair and pro-apoptotic factors in rat germinal cells exposed to nitric oxide donors by Silvia Di Meglio; Filomena Tramontano; Gabriella Cimmino; Roy Jones; Piera Quesada (35-44).
We describe the involvement of poly(ADP-ribose)polymerase 1 and 2 (PARP-1 and -2) and poly(ADP-ribose)glycohydrolase (PARG) in the response of rat germinal cells to the action of the NO donors, 3-morpholino-sydnonimine (SIN-1) and spermine nonoate (SNO). Primary spermatocytes and round spermatids showed a differential sensitivity to DNA damage induced by acute exposure to SIN-1 and SNO. Spermatocytes were able to repair DNA damage caused by the release of NO from SNO but neither spermatocytes nor spermatids could recover from the release of NO and O2 •− from SIN-1. Addition of the PARPs inhibitor, 3-aminobenzamide, and the PARG inhibitor, gallotannin (GT), to germ cell cultures impaired DNA repair significantly. Consistent with the DNA repair seen in primary spermatocytes, both SIN-1 and SNO induced PARPs activation in these cells. In the case of SIN-1, there was an immediate but transient response while SNO induced a delayed but more sustained increase in PARPs activity. Chronic exposure of spermatocytes to SIN-1 and SNO, however, committed the cells to apoptosis, which coincided with proteolysis of PARP-1. The data indicate a dual role for PARPs and PARG in germinal cells as key proteins in processes that sense and repair DNA damage as well as in the commitment to apoptosis following prolonged oxidative stress.
Keywords: Poly(ADPR)polymerase 1 (PARP-1); Poly(ADPR)polymerase 2 (PARP-2); Poly(ADPR)glicohydrolase (PARG); NO donor; DNA repair; Apoptosis; Germinal cell;