Steroid hormone receptors activate specific gene transcription by binding as hormone-receptor complexes to DNA enhancer elements termed hormone responsive elements. A highly conserved 66-amino-acid region of the oestrogen and glucocorticoid receptors which corresponds to part of the receptor DNA-binding domain (region C) determines the specificity of target gene recognition. This region contains two subregions (CI and CII), encoded in two separate exons, that are analogous to the 'zinc fingers' of the transcription factor TFIIIA. The N-terminal CI finger determines the recognition specificity of the hormone responsive element. A chimaeric oestrogen receptor, in which the CI finger is replaced with the corresponding glucocorticoid receptor CI finger region, activates transcription from a reporter gene containing a glucocorticoid-responsive element, but not from a reporter gene containing an oestrogen-responsive element. We report here that three amino acids located at the C-terminal side of the oestrogen receptor CI finger play a key part in this specificity.
Hematopoietic stem and progenitor cells (HSPCs) are exposed to low levels of oxygen in the bone marrow niche, and hypoxia-inducible factors (HIFs) are the main regulators of cellular responses to oxygen variation. Recent studies using conditional knockout mouse models have unveiled a major role for HIF-1α in the maintenance of murine HSCs; however, the role of HIF-2α is still unclear. Here, we show that knockdown of HIF-2α, and to a much lesser extent HIF-1α, impedes the long-term repopulating ability of human CD34(+) umbilical cord blood cells. HIF-2α-deficient HSPCs display increased production of reactive oxygen species (ROS), which subsequently stimulates endoplasmic reticulum (ER) stress and triggers apoptosis by activation of the unfolded-protein-response (UPR) pathway. HIF-2α deregulation also significantly decreased engraftment ability of human acute myeloid leukemia (AML) cells. Overall, our data demonstrate a key role for HIF-2α in the maintenance of human HSPCs and in the survival of primary AML cells.
Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder that is associated with an inherited defect of the nucleotide excision repair pathway (NER). In this study, we investigated the involvement of XP genes in 86 XP patients belonging to 66 unrelated families, most of them consanguineous and originating from Maghreb. Sequencing analysis was performed either directly (44 probands) or after having previously characterized the involved XP gene by complementation assay (22 families). XPC and XPA mutations were respectively present in 56/66 and 8/66 probands. Strikingly, we identified the same homozygous frameshift mutation c.1643_1644delTG (p.Val548AlafsX25) in 87% of XP-C patients. Haplotype analysis showed a common founder effect for this mutation in the Mediterranean region, with an estimated age of 50 generations or 1,250 years. Among 7/8 XP-A patients, we found the previously reported nonsense homozygous XPA mutation (p.Arg228X). Six mutations--to our knowledge previously unreported--(five in XPC, one in XPA) were also identified. In conclusion, XPC appears to be the major disease-causing gene concerning xeroderma pigmentosum in North Africa. As the (p.Val548AlafsX25) XPC mutation is responsible for a huge proportion of XP cases, our data imply an obvious simplification of XP molecular diagnosis, at least in North Africa.
Besides lung, postnatal human epidermis is the only epithelium in direct contact with atmospheric oxygen. Skin epidermal oxygenation occurs mostly through atmospheric oxygen rather than tissue vasculature, resulting in a mildly hypoxic microenvironment that favors increased expression of hypoxia-inducible factor-1α (HIF-1α). Considering the wide spectrum of biological processes, such as angiogenesis, inflammation, bioenergetics, proliferation, motility, and apoptosis, that are regulated by this transcription factor, its high expression level in the epidermis might be important to HIF-1α in skin physiology and pathophysiology. Here, we review the role of HIF-1α in cutaneous angiogenesis, skin tumorigenesis, and several skin disorders.
UV-induced apoptosis in keratinocytes is a highly complex process in which various molecular pathways are involved. These include the extrinsic pathway via triggering of death receptors and the intrinsic pathway via DNA damage and reactive oxygen species (ROS) formation. In this study we investigated the effect of catalase and CuZn-superoxide dismutase (SOD) overexpression on apoptosis induced by UVB exposure at room temperature or 4°C on normal human keratinocytes. Irradiation at low temperature reduced UV-induced apoptosis by 40% in normal keratinocytes independently of any change in p53 and with a decrease in caspase-8 activation. Catalase overexpression decreased apoptosis by 40% with a reduction of caspase-9 activation accompanied by a decrease in p53. Keeping cells at low temperature and catalase overexpression had additive effects. CuZn-SOD overexpression had no significant effect on UVB-induced apoptosis. UVB induced an increase in ROS levels at two distinct stages: immediately following irradiation and around 3 h after irradiation. Catalase overexpression inhibited only the late increase in ROS levels. We conclude that catalase overexpression has a protective role against UVB irradiation by preventing DNA damage mediated by the late ROS increase.
Porphobilinogen deaminase (hydroxymethylbilane synthase; EC 4.3.1.8), the third enzyme of the heme biosynthetic pathway, catalyzes the stepwise condensation of four porphobilinogen units to yield hydroxymethylbilan, which is in turn converted to uroporphyrinogen III by cosynthetase. We compared the apparent molecular mass of porphobilinogen deaminase from erythropoietic and from non‐erythropoietic cells by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immune‐blotting. The results indicate that two isoforms of porphobilinogen deaminase can be distinguished and differ by 2000 Da. Analysis of cell‐free translation products directed by mRNAs from human erythropoietic spleen and from human liver demonstrates that the two isoforms of porphobilinogen deaminase are encoded by distinct messenger RNAs. We cloned and sequenced cDNAs complementary to the non‐erythropoietic form of porphobilinogen deaminase encoding RNA. Comparison of these sequences to that of human erythropoietic mRNA [Raich et al. (1986) Nucleic Acids Res. 14, 5955–5968] revealed that the two mRNA species differ by their 5′ extremity. From the mRNA sequences we could deduce that an additional peptide of 17 amino acid residues at the NH2 terminus of the non‐erythropoietic isoform of porphobilinogen deaminase accounts for its higher molecular mass. RNase mapping experiments demonstrate that the two porphobilinogen deaminase mRNAs are distributed according to a strict tissue‐specificity, the erythropoietic form being restricted to erythropoietic cells. We propose that a single porphobilinogen deaminase gene is transcribed from two different promoters, yielding the two forms of porphobilinogendeaminase mRNAs. Our present finding may have some relevance for further understanding the porphobilinogen deaminase deficiency in certain cases of acute intermittent porphyria with an enzymatic defect restricted in non‐erythropoietic cells.
The JAK2V617F somatic point mutation has been described in patients with myeloproliferative disorders (MPDs). Despite this progress, it remains unknown how a single JAK2 mutation causes 3 different MPD phenotypes, polycythemia vera (PV), essential thrombocythemia, and primitive myelofibrosis (PMF). Using an in vivo xenotransplantation assay in nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice, we tested whether disease heterogeneity was associated with quantitative or qualitative differences in the hematopoietic stem cell (HSC) compartment. We show that the HSC compartment of PV and PMF patients contains JAK2V617F-positive longterm, multipotent, and self-renewing cells. However, the proportion of JAK2V617F and JAK2 wild-type SCID repopulating cells was dramatically different in these diseases, without major modifications of the selfrenewal and proliferation capacities for JAK2V617F SCID repopulating cells. These experiments provide new insights into the pathogenesis of JAK2V617F MPD and demonstrate that a JAK2 inhibitor needs to target the HSC compartment for optimal disease control in classical MPD.
Hypoxia-inducible factor-1 (HIF-1) is a major transcription factor sensitive to oxygen levels, which responds to stress factors under both hypoxic and nonhypoxic conditions. UV irradiation being a common stressor of skin, we looked at the effect of UVB on HIF-1␣ expression in keratinocytes. We found that UVB induces a biphasic HIF-1␣ variation through reactive oxygen species (ROS) generation. Whereas rapid production of cytoplasmic ROS down-regulates HIF-1␣ expression, delayed mitochondrial ROS generation results in its up-regulation. Indeed, activation of p38 MAPK and JNK1 mediated by mitochondrial ROS were required for HIF-1␣ phosphorylation and accumulation after UVB irradiation. Our experiments also revealed a key role of HIF-1␣ in mediating UVB-induced apoptosis. We conclude that the broad impact of the HIF-1 transcription factor on gene expression could make it a key regulator of UV-responsive genes and photocarcinogenesis. Solar UV radiation is the major risk factor for the development of skin cancer, the most common malignancy in the world. Several factors participate in the human natural photoprotective barrier, including constitutive and UV-induced pigmentation, stratum corneum thickness, UV-induced immune responses, UV-induced apoptosis, antioxidant defense, and DNA repair systems (1, 2). To develop a strategy for the prevention and the therapy of skin cancer, relationships between the various cellular signaling pathways that modulate cellular responses to UV irradiation in skin cells require a more indepth understanding. Within this complex group of UV-induced cellular responses, the role of ROS 3 appears central with dual effects (3). On one hand, an increase in ROS concentrations following UVB irradiation leads to cytotoxicity through protein, lipid, and DNA oxidation both in vitro and in vivo (4 -7). Reduction of the deleterious effects of UV-induced ROS by reinforcement of the antioxidant defense supports this notion (8 -10). On the other hand, ROS can also act as second messenger molecules mediating the response of cells to UVB (11)(12)(13)(14). We have recently demonstrated that UVB induces ROS production in two distinct waves. Whereas the second peak in ROS concentration has a direct effect on apoptosis, the first peak could mediate signal transduction (10). In this study, we addressed the possible role of ROS as second messengers after UVB irradiation and their potential relationship with a stress-inducible transcription factor, HIF-1. This factor, so named for its exquisite sensitivity to oxygen levels in the cell environment, participates in the regulation of numerous genes involved in angiogenesis, glycolysis, apoptosis, migration, and metastasis (15-18). We decided to look at HIF-1␣ expression within the context of ROS production in keratinocytes, in response to UVB irradiation, because ROS were already known to influence HIF-1␣ regulation and hypoxia-induced apoptosis (19). Moreover, involvement of HIF in the modulation of cell responses to growth factors under normoxia in a ROS-dependen...
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