Impaired myelopoiesis in mice devoid of interferon regulatory factor 1.

Interferon regulatory factor (IRF)-1 is a transcription factor controlling the expression of several genes, which are differentially induced depending on the cell type and signal. IRF-1 modulates multiple functions, including regulation of immune responses and host defence, cell growth, cytokine signalling and hematopoietic development. Here, we investigated the role of IRF-1 in granulocytic differentiation in mice with a null mutation in the IRF-1 gene. We show that IRF-1(-/-) bone marrow cells exhibit an increased number of immature granulocytic precursors, associated with a decreased number of mature granulocytic elements as compared to normal mice, suggestive of a defective maturation process. Clonogenetic analyses revealed a reduced number of CFU-G, CFU-M and CFU-GM colonies in IRF-1(-/-) mice, while the number of BFU-E/CFU-E colonies was unchanged. At the molecular level, the expression of CAAT-enhancer-binding protein (C/EBP)-epsilon, -alpha and PU.1 was substantially lower in the CD11b(+) cells from the bone marrow of IRF-1(-/-) mice as compared to cells from wild-type mice. These results, together with the fact that IRF-1 is markedly induced early during granulo-monocytic differentiation of CD34+ cells, highlight the pivotal role of IRF-1 in the early phases of myelopoiesis.

Activation and repression of the 2-5A synthetase and p21 gene promoters by IRF-1 and IRF-2.

The Interferon Regulatory Factors-1 and -2 (IRF-1 and IRF-2) were originally identified as transcriptional regulators of the interferon (IFN) and IFN-stimulated genes. These factors also modulate immune response and play a role in cell growth regulation. In this study we analysed the effect of the ectopic expression of IRF-1 and IRF-2 on the regulation of two potential IRF target genes involved in cell growth regulation, 2-5A synthetase and p21 (WAF/CP1), both of which contain consensus binding sites for IRF family members within their promoters. Following ectopic expression, IRF-1 transactivated 2-5A synthetase and p21 genes, an effect that was counterbalanced by concomitant ectopic expression of IRF-2. These effects were mediated by direct binding of IRF to the gene promoters. A construct expressing an IRF-2 antisense (FRI-2) was able to revert the inhibitory effect of IRF-2 on the IRF-1 transactivation. IRF-1 also induced expression of its homologous repressor IRF-2 as indicated by EMSA analysis using an IRF-E probe from the IRF-2 promoter; and by cotransfection of IRF-1 together with an IRF-2 promoter CAT construct. Therefore, the induction of IRF-1 by IFNs or other stimuli acts as a transactivator of genes involved in cell growth regulation, as well as of its own repressor IRF-2, thus providing autoinhibitory regulation of IRF-1 activated genes.

Regulation of expression of ferritin H-chain and transferrin receptor by protoporphyrin IX.

The effect of protoporphyrin IX (hemin without iron) on the expression of transferrin receptor and ferritin was investigated in Friend leukemia cells. Cells treated with protoporphyrin IX exhibit enhanced transferrin-receptor expression and markedly reduced ferritin synthesis. Stimulation of transferrin-receptor expression is observed at both the mRNA and protein level. The effect on ferritin synthesis is mediated by translational inhibition of the mRNA, which, in contrast, is transcriptionally stimulated by protoporphyrin IX treatment. The regulation of transferrin receptor and ferritin in response to iron perturbations has been studied extensively and is mediated by the binding of iron-regulatory proteins (IRP) to the iron-responsive elements (IRE) present in the 3' and 5' untranslated regions of the transferrin-receptor and ferritin mRNA, respectively. To elucidate the molecular mechanisms underlying the effects of protoporphyrin IX on ferritin and transferrin-receptor expression, the role of the IRE sequence was investigated both in vivo by transfection experiments, with a construct containing the coding region for the chloramphenicol acetyltransferase (CAT) reporter gene under the translational control of the ferritin IRE, and in vitro by RNA band-shift assays. Whereas, examination of IRP binding to the IRE by in vitro assays suggests an apparent inactivation of IRP by protoporphyrin IX treatment, CAT assays indicate that protoporphyrin IX is able to induce in vivo a translational inhibition similar to that obtained by treatment with the iron chelator Desferal. This observation raises the possibility of different effects on the IRP activity exerted by porphyrin treatment in intact tissue-culture cells and in vitro. We conclude that translation of ferritin mRNA and degradation of transferrin-receptor mRNA are inhibited in intact tissue-culture cells by protoporphyrin IX through a mechanism similar to that exerted by iron chelation, thus involving depletion of the intracellular iron pool. These results can improve the understanding of the regulation of ferritin gene expression in some pathological conditions associated with disturbed heme synthesis.

Regulation of ferritin H-chain expression in differentiating Friend leukemia cells.

The mechanisms that regulate the expression of ferritin, the iron storage protein, have been investigated in Friend erythroleukemia cells (FLCs) induced to differentiate by several chemical compounds. In differentiating FLCs, administration of hemin increases the steady-state level of ferritin mRNA about 15-fold and the ferritin content about 20- to 25-fold. Conversely, iron salts have only mild stimulatory effects on these parameters and iron chelators only slightly inhibited the stimulatory effect of hemin. Transient transfection experiments with a construct in which the human ferritin H-chain promoter drives the expression of the indicator chloramphenicol acetyltransferase (CAT) gene show that the increase in mRNA content is mainly due to enhanced transcription. In addition to transcriptional effects, translational regulation resulting in the further increase in ferritin synthesis is shown by CAT assays from cells transiently transfected with a construct containing the coding region for the indicator CAT mRNA under the translational control of the mRNA ferritin iron-responsive element. We conclude that, in FLCs induced to differentiate, hemin acts synergistically with the differentiation inducers, increasing ferritin expression. Both transcriptional and translational mechanisms are responsible for this synergistic effect, which appears to be characteristic of differentiated erythroid cells because it is not observed in other cell types (ie, fibroblastic cell lines).

Cells resistant to interferon-beta respond to interferon-gamma via the Stat1-IRF-1 pathway.

The mechanism responsible for the induction of the 2-5A synthetase gene by Interferon-gamma (IFN-gamma) (type II) was studied in Friend leukemia cells. It was previously shown that activation of 2-5A synthetase gene expression by IFN-gamma in the 3Cl8 cell, a clone resistant to IFN-alpha,beta (type I), correlates with the formation of two major complexes, designated Fg and Fc, that bind to the interferon-stimulated responsive element of the gene. Conversely, in a clone resistant to both types of IFNs (3 gamma R8), no induction of DNA-protein complexes or of 2-5A synthetase gene expression was detected. In the present report the Fg complex has been characterized as including the interferon regulatory factor 1 (IRF-1), whereas the Fc factor, present also in control cells, has been characterized as composed of IRF-2. Incubation of cell extracts with antibodies to IRF-1 abolishes the formation of the Fg complex, and antibodies to IRF-2 abolish the formation of the Fc complex. Moreover, in the 3Cl8 cell, IFN-gamma is able to induce in few minutes the formation of a complex between a DNA element identified as the IFN-gamma activation site (GAS), present on the IRF-1 gene promoter, and the STAT1 protein. These findings suggest that in cells resistant to type I IFN, IFN-gamma is able, through the activation of the STAT1 protein, to induce the expression of the IRF-1 factor which in turn seems to be sufficient to transactivate the 2-5A synthetase gene.

Protein binding to the interferon response enhancer correlates with interferon induction of 2'-5'-oligoadenylate synthetase in normal and interferon-resistant Friend cells.

The induction of transcription of the 2'-5'-oligoadenylate (2-5A) synthetase gene by type I (alpha/beta) and type II (gamma) interferons (IFNs) has been studied in wild-type (w.t.) and IFN-resistant Friend leukemia cells (FLC). Following IFN treatment, new complexes are formed in vitro between the IFN-responsive sequence (IRS) of the 2-5A synthetase gene and cellular proteins. Within minutes after IFN-alpha/beta addition to w.t. FLC, an IRS-protein complex, designated F1, is detected, as already observed in several human cell lines. In response to IFN-gamma, a novel complex, designated Fg, is observed in w.t. FLC. The Fg complex appears within 3 h, while an F1-like complex is faintly visible 10 to 24 h later. In the IFN-alpha/beta-resistant FLC, IFN-gamma induces only the Fg complex and fails to induce F1. Fg formation is correlated with the IFN-gamma-induced transcription of the 2-5A synthetase gene and the appearance of the corresponding enzymatic activity in both w.t. and IFN-alpha/beta-resistant FLC. These findings suggest that F1 and Fg represent two distinct effector complexes by which type I and type II IFNs, respectively, induce 2-5A synthetase.

Positive modulation of hemoglobin, heme, and transferrin receptor synthesis by murine interferon-alpha and -beta in differentiating Friend cells. Pivotal role of heme synthesis.

Administration of highly purified preparations of murine interferon (IFN)-alpha 1, -alpha 4, -alpha 6, or -beta to Friend leukemia cells induced to differentiate by dimethyl sulfoxide leads to a 100% increase of benzidine-positive (B+) cells. Different efficiencies for the two IFN species have been observed; a 10-fold higher dose of IFN-alpha is needed for stimulation of hemoglobin production and inhibition of cell growth as compared with IFN-beta. Both species of IFN induce a substantial increase in heme, hemoglobin, and transferrin receptor levels. In vitro run-on transcription assays indicate that IFN-beta moderately stimulates transcription of the alpha-globin gene but not the transferrin receptor gene. It is postulated that IFN induces the enhancing effect on differentiation via a marked increase of heme synthesis and number of transferrin receptors, which in turn leads to an enhancement of globin chain synthesis. In this regard, the negative feedback reported in a variety of other cell types for the regulation of transferrin receptor expression by heme does not seem to be operative in maturing Friend erythroleukemia cells, which present evidence for a positive mechanism.

A full-length murine 2-5A synthetase cDNA transfected in NIH-3T3 cells impairs EMCV but not VSV replication.

Treatment of cells with interferons (IFNs) induces resistance to virus infection. The 2'-5'oligo A (2-5A) synthetase/RNase L is one of the pathways leading to translation inhibition induced by IFN treatment. A murine cDNA encoding the 43-kDa 2-5A synthetase was cloned and sequenced. NIH-3T3 cell clones transfected with this cDNA expressed the enzymatic activity to various extents and exhibited resistance to encephalomyocarditis virus (EMCV) but not to vesicular stomatitis virus replication. The specific resistance to EMCV can be attributed to 2-5A synthetase.