Caspase-3 plays a central role in apoptosis. It is also activated in normal erythropoiesis, with its activity peaking early during development (erythroid colonyforming unit [CFU-E] stage). In the present study, we have reduced the expression and subsequent enzymatic activity of caspase-3 by transfection of small interfering RNA (siRNA) directed to caspase-3 in a differentiating human erythroid culture system. We find that siRNA treatment yields a 50% reduction in cells that undergo enucleation with no change in the fraction of cells that undergo apoptosis, measured throughout the culture. Furthermore, a substantial fraction of treated cells are unable to complete the transition from pronormoblasts to basophilic normoblasts. These results demonstrate that caspase-3 is required for efficient erythropoiesis in this model system. IntroductionMembers of the caspase family of aspartate-specific proteases have been found to be essential in a variety of cells and tissues for differentiation and homeostasis, by virtue of their role in causing apoptotic cell death. Due to its potent effect on cell viability, caspase activity is tightly regulated. These proteins are expressed as inactive proenzymes that must be proteolytically cleaved into large and small fragments, which then associate as a tetramer to form a catalytically active enzyme. 1 Caspase activation proceeds by a cascade mechanism. External stimuli, such as death receptor (Fas, tumor necrosis factor-␣ [TNF-␣]) stimulation or cytokine withdrawal, and internal stimuli, such as genotoxic stress or mitochondrial permeabilization, activate a subset of the initiator caspases, 2, 8, 9, and 10. These cleave the effector caspases, 3, 6, and 7, which in turn cleave additional caspases and vital cellular targets. 1 Caspase substrates include structural proteins (actin, lamin B, and gelsolin 2,3 ), proteins required for DNA repair (poly(ADPribose) polymerase [PARP] 4 and DNA-dependent protein kinase 5 ), and proteins with specific apoptotic function (DNA fragmentation factor that releases caspase-activated DNase 6,7 ). Although there are distinct differences, 8 the striking similarities between programmed cell death and late stages of erythropoiesis have been noted 9,10 and include nuclear and chromatin condensation; cleavage of nuclear proteins, such as acinus, lamin B, and PARP; and possibly, caspase activation. However, the significance of these similarities, especially with respect to caspase activation, is not fully established.Erythropoiesis is a complex multistage process encompassing the differentiation of pluripotent hematopoietic progenitor cells to mature erythrocytes. The earliest cell committed to the erythroid lineage is the erythroid burst-forming unit (BFU-E). These then become erythroid colony-forming unit (CFU-E) cells, which are followed in turn by the morphologically distinguishable stages of pronormoblast and then basophilic, polychromatic, and orthochromatic normoblast. A key event of late-stage erythropoiesis is nuclear condensation followed by extr...
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The essential metals iron, zinc and copper deposit near the Aβ (amyloid β-peptide) plaques in the brain cortex of AD (Alzheimer’s disease) patients. Plaque-associated iron and zinc are in neurotoxic excess at 1 mM concentrations. APP (amyloid precursor protein) is a single transmembrane metalloprotein cleaved to generate the 40-42-amino-acid Aβs, which exhibit metal-catalysed neurotoxicity. In health, ubiquitous APP is cleaved in a non-amyloidogenic pathway within its Aβ domain to release the neuroprotective APP ectodomain, APP(s). To adapt and counteract metal-catalysed oxidative stress, as during reperfusion from stroke, iron and cytokines induce the translation of both APP and ferritin (an iron storage protein) by similar mechanisms. We reported that APP was regulated at the translational level by active IL (interleukin)-1 (IL-1-responsive acute box) and IRE (iron-responsive element) RNA stem-loops in the 5′ untranslated region of APP mRNA. The APP IRE is homologous with the canonical IRE RNA stem-loop that binds the iron regulatory proteins (IRP1 and IRP2) to control intracellular iron homoeostasis by modulating ferritin mRNA translation and transferrin receptor mRNA stability. The APP IRE interacts with IRP1 (cytoplasmic cis-aconitase), whereas the canonical ferritin-H IRE RNA stem-loop binds to IRP2 in neural cell lines, and in human brain cortex tissue and in human blood lysates. The same constellation of RNA-binding proteins [IRP1/IRP2/poly(C) binding protein] control ferritin and APP translation with implications for the biology of metals in AD.
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