In mouse embryonic fibroblasts, neither caspase-8 nor cellular FLICE-inhibitory protein (FLIP) is necessary for TNF to activate NF-κB, but caspase-8 is required for TNF to cause cell death, and induction of FLIP by NF-κB is required to prevent it.

Binding of TNF to TNF receptor-1 can give a pro-survival signal through activation of p65/RelA NF-κB, but also signals cell death. To determine the roles of FLICE-inhibitory protein (FLIP) and caspase-8 in TNF-induced activation of NF-κB and apoptosis, we used mouse embryonic fibroblasts derived from FLIP and caspase-8 gene-deleted mice, and treated them with TNF and a smac-mimetic compound that causes degradation of cellular inhibitor of apoptosis proteins (cIAPs). In cells treated with smac mimetic, TNF and Fas Ligand caused wild-type and FLIP(-/-) MEFs to die, whereas caspase-8(-/-) MEFs survived, indicating that caspase-8 is necessary for death of MEFs triggered by these ligands when IAPs are degraded. By contrast, neither caspase-8 nor FLIP was required for TNF to activate p65/RelA NF-κB, because IκB was degraded, p65 translocated to the nucleus, and an NF-κB reporter gene activated normally in caspase-8(-/-) or FLIP(-/-) MEFs. Reconstitution of FLIP(-/-) MEFs with the FLIP isoforms FLIP-L, FLIP-R, or FLIP-p43 protected these cells from dying when treated with TNF or FasL, whether or not cIAPs were depleted. These results show that in MEFs, caspase-8 is necessary for TNF- and FasL-induced death, and FLIP is needed to prevent it, but neither caspase-8 nor FLIP is required for TNF to activate NF-κB.

Puma indirectly activates Bax to cause apoptosis in the absence of Bid or Bim.

Bcl-2 family members regulate apoptosis in response to cytokine withdrawal and a broad range of cytotoxic stimuli. Pro-apoptotic Bcl-2 family members Bax and Bak are essential for apoptosis triggered by interleukin-3 (IL-3) withdrawal in myeloid cells. The BH3-only protein Puma is critical for initiation of IL-3 withdrawal-induced apoptosis, because IL-3-deprived Puma(-/-) cells show increased capacity to form colonies when IL-3 is restored. To investigate the mechanisms of Puma-induced apoptosis and the interactions between Puma and other Bcl-2 family members, we expressed Puma under an inducible promoter in cells lacking one or more Bcl-2 family members. Puma rapidly induced apoptosis in cells lacking the BH3-only proteins, Bid and Bim. Puma expression resulted in activation of Bax, but Puma killing was not dependent on Bax or Bak alone as Puma readily induced apoptosis in cells lacking either of these proteins, but could not kill cells deficient for both. Puma co-immunoprecipitated with the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1 but not with Bax or Bak. These data indicate that Puma functions, in the context of induced overexpression or IL-3 deprivation, primarily by binding and inactivating anti-apoptotic Bcl-2 family members.

Caspase inhibitors: viral, cellular and chemical.

Caspases, key mediators of apoptosis, are a structurally related family of cysteine proteases that cleave their substrates at aspartic acid residues either to cause cell death or to activate cytokines as part of an immune response. They can be controlled upstream by the regulation of signals that lead to zymogen activation, or downstream by inhibitors that prevent them from reaching their substrates. This review specifically looks at caspase inhibitors as distinct from caspase regulators: those produced by the cell itself; those whose genes are carried by viruses; and artificial caspase inhibitors used for research and potentially as therapeutics.

Hydrophobic residues Phe751 and Leu753 are essential for STAT5 transcriptional activity.

One facet of cytokine signaling is relayed to the nucleus by the activation, through tyrosine phosphorylation, of latent cytoplasmic signal transducers and activators of transcription (STAT) family members. It has been demonstrated that the C termini of STATs contain the transactivation domain and are essential for the transactivation of target genes. To better understand the function of the STAT C terminus, we have generated a series of C-terminal mutants in STAT5a and examined their effects on transactivation, tyrosine phosphorylation, and DNA binding. Using GAL4 chimerae with the C terminus of STAT5, we have defined a 12-amino acid region essential for STAT5 transactivation. Surprisingly, deletion of these 12 amino acids in the context of the native STAT5 backbone preserved the overall transcriptional activity of the protein. Further analysis revealed that deletion of this region resulted in hyper-DNA binding activity, thus compensating for the weakened transactivation domain. Using site-directed mutagenesis, we show that within this 12-amino acid region the acidic residues were non-essential for transactivation. In contrast, the non-acidic residues were crucial for transactivation. Mutating either Phe(751) or Leu(753) to alanine abolished transactivation suggesting that these residues were essential for connecting STAT5 to the basal transcriptional machinery.

Rapid selection of tetracycline-controlled inducible cell lines using a green fluorescent-transactivator fusion protein.

We describe a modification of the tetracycline-controlled expression system that facilitates the rapid identification of tetracycline-sensitive clones. The TetR/VP16 transactivator protein was tagged with the green fluorescent protein (GFP) at its N-terminus. This results in a functional transactivator which allows cells expressing high levels of the modified transactivator to be selected by fluorescent-activated cell sorting. After expansion, single cell clones that maintain a high level of GFP fluorescence can be tested for their ability to transactivate a luciferase gene under control of the Tet operator, leading to the rapid identification of clones with strong inducibility.

Interleukin-3-induced activation of the JAK/STAT pathway is prolonged by proteasome inhibitors.

One facet of cytokine receptor signaling involves the activation of signal transducers and activators of transcription (STATs). STATs are rapidly activated via tyrosine phosphorylation by Janus kinase (JAK) family members and subsequently inactivated within a short period. We investigated the effect of proteasome inhibition on interleukin-3 (IL-3) activation of the JAK/STAT pathway following stimulation of Ba/F3 cells. Treatment of Ba/F3 cells with the proteasome inhibitor, N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL), led to stable tyrosine phosphorylation of the IL-3 receptor, beta common (betac), and STAT5 following stimulation. The effects of LLnL were not restricted to the JAK/STAT pathway, as Shc and mitogen-activated protein kinase (MAPK) phosphorylation were also prolonged in LLnL-treated cells. Further investigation showed these stable phosphorylation events were the result of prolonged activation of JAK2 and JAK1. These observations were confirmed using pharmacologic inhibitors. In the presence of LLnL, stable phosphorylation of STAT5 and betac was abrogated if the tyrosine kinase inhibitor, staurosporine, was added. The effect of staurosporine on STAT5 phosphorylation could be overcome if the phosphatase inhibitor, vanadate, was also added, suggesting phosphorylated STAT5 could be stabilized by phosphatase, but not by proteasome inhibition per se. These observations are consistent with the hypothesis that proteasome-mediated protein degradation can modulate the activity of the JAK/STAT pathway by regulating the deactivation of JAK.

Complex regulation of transferrin receptors during erythropoietin-induced differentiation of J2E erythroid cells--elevated transcription and mRNA stabilisation produce only a modest rise in protein content.

The regulation of transferrin-receptor synthesis was studied in J2E erythroid cells induced to differentiate with erythropoietin. Nuclear run-on assays demonstrated that transcription of the transferrin-receptor gene rose markedly after erythropoietin treatment. In addition, transferrin-receptor mRNA was stabilised and this was associated with an increase in the activity of the RNA-binding protein IRP (iron regulatory protein). As a result of increased transcription and mRNA stabilisation, steady-state RNA levels increased 10-20-fold. However, despite these large increases in mRNA, translation only doubled; consequently, modest increases in total protein and surface transferrin receptors were observed. Moreover, this rise in transferrin receptors was transient, and correlated with a burst of proliferation shortly after erythropoietin treatment. The expected inverse relationship between transferrin receptors and ferritin did not occur during J2E maturation as translation of both ferritin subunits increased when transferrin-receptor mRNA levels rose. Analysis of mutant J2E clones incapable of synthesising haemoglobin revealed that surface transferrin-receptor levels were only 15-25% that of the parental erythroid line. We propose that the surface expression of transferrin receptors in J2E cells is governed by three factors: basal levels essential for normal growth in culture; elevated levels needed for haemoglobin synthesis; and a transient erythropoietin-induced increase that is required for the final burst of proliferation. It was concluded that the regulation of transferrin-receptor production in erythropoietin-stimulated J2E cells is complex and that there are several sites of control.

Haemoglobin synthesis in erythropoietin-stimulated J2E cells does not require increased numbers of transferrin receptors.

Changes in transferrin-receptor numbers and iron utilisation were monitored during erythropoietin-induced maturation of J2E erythroid cells. Uptake of transferrin and iron doubled 24 h after exposure to erythropoietin, due to a twofold rise in surface transferrin receptors. In addition, a tenfold increase in iron incorporation into haem was observed after erythropoietin stimulation, as iron taken up from transferrin was directed towards haem biosynthesis and away from storage in ferritin. The rise in iron chelation into haem correlated extremely well with haemoglobin synthesis. However, the increase in numbers of transferrin receptors was not essential for haemoglobin synthesis; rather, it was linked with a burst in proliferation stimulated by erythropoietin. We have shown previously that amiloride blocks erythropoietin-enhanced proliferation of J2E cells, but potentiates maturation [Callus, B. A., Tilbrook, P. A., Busfield, S. J. & Klinken, S. P. (1995) Exp. Cell Res. 219, 39-46]. Here we demonstrate that amiloride suppressed the hormone-induced increase in transferrin receptors, whereas the enhanced incorporation of iron into haem was not inhibited. Similarly, when sodium butyrate was used to induce differentiation of J2E cells, proliferation ceased and surface transferrin receptors remained unaltered, while haemoglobin production was accelerated. It was concluded from these experiments that the erythropoietin-stimulated rise in transferrin receptors during the final stages of J2E cell maturation is linked to cell division, and is not essential for haemoglobin synthesis.

Effects of overexpression of the transferrin receptor on the rates of transferrin recycling and uptake of non-transferrin-bound iron.

The possibilities that the recycling of the transferrin receptor is a rate-limiting step in the efflux of endocytosed transferrin, and that the receptor functions as a trans-membrane Fe transporter were investigated in untransfected Ltk- cells and in cells transfected with different levels of DNA for wild-type, mutant and chimeric human transferrin receptors. The uptake of transferrin-bound Fe and non-transferrin-bound Fe(II), and the surface binding, endocytosis and recycling of transferrin were measured. In cells that expressed increasing numbers of surface transferrin receptors, the rate of Fe uptake increased at a slower rate than the number of receptors. By measurement of the rates of endocytosis and recycling of transferrin it was shown that this effect was not due to a deficiency of endocytosis, but to a slower rate of recycling as the receptor numbers increased. Hence, a restricted recycling rate of the transferrin receptor appeared to be responsible for the slower rate of Fe uptake by cells with high receptor numbers, presumably because one or more cytosolic components required for recycling were in limited supply. The rate of uptake of non-transferrin-bound Fe(II) was not influenced by the number of transferrin receptors present on the surface of the cells even though this varied more than 20-fold between the different cell lines. Hence, this investigation does not support the hypothesis that the receptors play a direct role in the transport of Fe(II) across cell membranes, as has been proposed previously [Singer, S. J. (1989) Biol. Cell 65, 1-5].

Regulation of the erythropoietin receptor and involvement of JAK2 in differentiation of J2E erythroid cells.

In response to erythropoietin, J2E cells proliferate and differentiate into mature hemoglobin-producing erythroid cells. Here we show that following hormonal stimulation, between 10 and 17 proteins, including the erythropoietin receptor and JAK2, were tyrosine phosphorylated immediately after exposure to the hormone. Although the receptor was only phosphorylated to 15% of its maximum with 0.1 unit/ml erythropoietin, this was sufficient to induce peak hemoglobin synthesis. The importance of JAK2 to J2E cell maturation was demonstrated by inhibiting JAK2 protein synthesis with antisense oligonucleotides; not only was erythropoietin-stimulated mitogenesis inhibited by this procedure, but differentiation was also suppressed. In addition, the activation of STAT5 paralleled the kinetics of receptor phosphorylation. During differentiation, 94% decrease in surface erythropoietin receptors was detected 48 h after ligand binding, but transcription of the receptor gene, mRNA steady-state levels, protein content, and translation rates did not alter with hormonal stimulation. We concluded from these experiments that (a) sub-maximal receptor phosphorylation is sufficient for differentiation to proceed; (b) JAK2 is required for erythropoietin-induced cell division and maturation; and (c) post-translational processing, or translocation, play important roles in controlling surface erythropoietin receptor numbers.

Erythropoietin exerts transcriptional and translational control over globin synthesis in J2E cells.

The J2E erythroid cell line, generated by transforming fetal liver cells, terminally differentiates in response to erythropoietin (epo). The cells expressed both adult and embryonic globin genes, although considerably more adult globin was produced, and transcripts for both species rose following exposure to epo. A 6-fold increase in transcription of the adult alpha and beta maj globin genes was observed after hormonal stimulation, which resulted in a substantial accumulation of mRNA. In addition, a modest but transient rise in translation enabled a 6-fold elevation in globin protein to occur. Concurrently, the total heme content rose markedly, enhancing hemoglobin synthesis 10-fold. The prosthetic group complexed entirely with globin proteins, and the hemoglobin produced was present as fully functional oxyhemoglobin, capable of gaseous exchange. We concluded, therefore, that hemoglobin synthesis in epo-induced J2E cells normally results from the coordinate stimulation of heme and globin synthesis. However, some mutant clones emerged where concomitant increases in globin and heme were not observed. Despite similar profiles for the appearance of hemoglobin and equivalent amounts of the oxygen carrier, several noticeable differences in globin synthesis were detected between epo-induced J2E cells and DMSO-stimulated murine erythroleukemia cells, i.e., the types of globin genes expressed, patterns of mRNA and protein production, and translation rates. These results demonstrate that the J2E cells provide a useful model system for investigating the molecular mechanisms of epo-initiated hemoglobin synthesis.

Diaminofluorene is more sensitive than benzidine for detecting hemoglobin in erythropoietin responsive J2E cells.

We have used the diaminofluorene stain to detect hemoglobin production in J2E cells following erythropoietin-induced differentiation. The pseudo-peroxidase activity of hemoglobin produces a colored product, fluorene blue, which can be measured spectrophotometrically. We found that the absorbance varied with time and concentration of hemoglobin, making it unsuitable for rapid, routine use. However, hemoglobin content could be determined from the initial reaction rate and this correlated extremely well with the number of benzidine positive cells. When used as a direct cytochemical stain diaminofluorene was shown to be more sensitive than benzidine in detecting hemoglobin-producing J2E cells.