A phosphorothioate‐oligonucleotide‐based antisense strategy for depleting MAP kinase was developed. The 17mer antisense probe, EAS 1, caused a potent and concentration‐dependent decrease in the steady state expression of p42 and p44 MAP kinase in 3T3 L1 fibroblasts and adipocytes with submicromolar concentrations effective. Antisense EAS 1 elicited a dose‐dependent inhibition of insulin‐ and serum‐stimulated DNA synthesis. Elimination of p42 MAP kinase by > 95% and p44 MAP kinase to levels undetected blocked the ability of serum in 3T3 L1 fibroblasts and insulin in 3T3 L1 adipocytes to stimulate DNA synthesis by 87‐95%. The differentiation of 3T3 L1 fibroblasts into adipocytes was prevented by 1 microM antisense EAS 1. The corresponding sense, scrambled or sense plus antisense EAS 1 phosphorothioate oligonucleotides did not deplete the p42 or p44 MAP kinase from either cell type, did not inhibit stimulation of DNA synthesis and did not interfere with differentiation. Two kinases on different MAP kinase activation pathways were not depleted by antisense EAS 1 whereas the ability of insulin to activate p90 S6 kinase was > 90% eliminated in 3T3 L1 adipocytes by 4.5 microM antisense EAS 1. In conclusion these results show that MAP kinase is required for insulin and serum stimulation of DNA synthesis, for insulin stimulation of p90 S6 kinase activity and for differentiation of 3T3 L1 cells. Moreover, the development of the antisense probe EAS 1 against a target sequence of p42 MAP kinase that is conserved in p44 MAP kinase and across a range of species provides a molecular tool of general applicability for further dissecting the precise targets and roles of MAP kinase.
Nramp1 (natural resistance-associated macrophage protein) was positionally cloned as the defective biallelic locus in inbred mouse strains associated with uncontrolled proliferation of obligate intracellular macrophage pathogens. The causative defect was described as G169D within membrane spanning domain 4 of a transporter. The biochemical activity of Nramp1 is implied from sequence conservation with Nramp2. Nramp2 encodes a divalent cation transporter and is the carrier of a defect in models of microcytic anaemia, associated with impaired intestinal iron uptake. Iron sequestration has been proposed as an antimicrobial mechanism. Therefore, such an activity for Nramp1 is consistent with model systems. Here we showed that Nramp1 directs iron transport within the macrophage. We describe stable, high-level Nramp1G169 allele-derived polypeptide expression in Balb/c Nramp1D169 RAW264.7 cells. Transfectants express levels, comparable to those in Nramp1G169-resistant macrophages, of a 90-100x103 MW Nramp1 polypeptide. Expression of the Nramp1 polypeptide correlates with lower cellular iron loads and a reduced chelatable iron pool following challenge with iron: nitrilotriacetate. Pulse chase experiments support an enhanced iron flux in expressing cells. These data are supported using the fluorescent iron probe calcein. In Nramp1G169-expressing cells we observed an increased iron flux into the cytoplasm from a calcein-inaccessible cellular location. These data suggest Nramp1, in resting macrophage cells, mobilizes iron, from an intracellular vesicle, which is destined for cell secretion. We propose that under these conditions Nramp1 plays a role in a salvage pathway of iron recycling.
Nramp1 (natural resistance-associated macrophage protein) controls innate immunity and encodes a transporter of unknown function. Here we describe an antibody to Nramp1 displaying immunoreactivity towards a mature heavily glycosylated polypeptide of 90^100 kDa and a precursor form of 45 kDa in macrophages. Ectopic expression of the Nramp1 cDNA in COS-1 cells demonstrates that Nramp1 modulates cellular iron levels following loading with low molecular weight iron chelates. Surprisingly, Nramp1 does not enhance iron uptake, but expression is associated with reduced cellular iron loads. We propose Nramp1 may play a role in a salvage pathway of iron recycling.z 1998 Federation of European Biochemical Societies.
Recent studies have identified a limited number of cellular receptors that can stimulate an alternative NF-B activation pathway that depends upon the inducible processing of NF-B2 p100 to p52. Here it is shown that the latent membrane protein (LMP)-1 of EpsteinBarr virus can trigger this signaling pathway in both B cells and epithelial cells. LMP1-induced p100 processing, which is mediated by the proteasome and is dependent upon de novo protein synthesis, results in the nuclear translocation of p52⅐RelB dimers. Previous studies have established that LMP1 also stimulates the canonical NF-B-signaling pathway that triggers phosphorylation and degradation of IB␣. Interestingly, LMP1 activation of these two NF-B pathways is shown here to require distinct regions of the LMP1 C-terminal cytoplasmic tail. Thus, C-terminal-activating region 1 is required for maximal triggering of p100 processing but is largely dispensable for stimulation of IB␣ phosphorylation. In contrast, C-terminal-activating region 2 is critical for maximal LMP1 triggering of IB␣ phosphorylation and up-regulation of p100 levels but does not contribute to activation of p100 processing. Because p100 deletion mutants that constitutively produce p52 oncogenically transform fibroblasts in vitro, it is likely that stimulation of p100 processing by LMP1 will play an important role in its transforming function.
Nramp1 (natural resistance-associated macrophage protein one) regulates intracellular pathogen proliferation and macrophage inflammatory responses. Murine Nramp1 exhibits a natural polymorphism with alleles termed resistant and susceptible. Alleles restrict or allow the proliferation of intracellular pathogens, respectively. Structural predictions suggest that Nramp1 encodes the prototypic member of a transporter family. Nramp1 exhibits sequence identity to Nramp2, which regulates intestinal and reticulocyte iron uptake. Based on this sequence identity we have initiated experiments for Nramp1 to investigate its role in macrophage iron homoeostasis and using a transfection approach in the RAW264.7 murine macrophagelike cell line, which lacks a functional Nramp1 gene. Nramp1 expression supports increased acute cytoplasmic influx of iron, detected using the fluorescent iron sensor dye calcein. Analysis of the endogenous iron sensors, iron regulatory protein 1 and 2, reveals a greater flux of iron in Nramp1-expressing cells and in its exclusion from the cytoplasm. Other work supports the prediction that Nramp1 is a phosphoprotein and the extent of phosphorylation changes in response to inflammatory cytokines. Together these data support the hypothesis that control of intracellular iron homoeostasis is a vital element used by phagocytes to control the proliferation of intracellular pathogens.
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