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Splenectomy improves the clinical parameters of patients with hereditary spherocytosis, but its potential benefit to red blood cell (RBC) functionality and the mechanism behind this benefit remain largely overlooked. Here, we compared 7 nonsplenectomized and 13 splenectomized patients with mutations in the β-spectrin or the ankyrin gene. We showed that hematological parameters, spherocyte abundance, osmotic fragility, intracellular calcium, and extracellular vesicle release were largely but not completely restored by splenectomy, whereas cryohemolysis was not. Affected RBCs exhibited decreases in β-spectrin and/or ankyrin contents and slight alterations in spectrin membrane distribution, depending on the mutation. These modifications were found in both splenectomized and nonsplenectomized patients and poorly correlated with RBC functionality alteration, suggesting additional impairments. Accordingly, we found an increased abundance of septins, small guanosine triphosphate-binding cytoskeletal proteins. Septins-2, -7, and -8 but not -11 were less abundant upon splenectomy and correlated with the disease severity. Septin-2 membrane association was confirmed by immunolabeling. Except for cryohemolysis, all parameters of RBC morphology and functionality correlated with septin abundance. The increased septin content might result from RBC maturation defects, as evidenced by (1) the decreased protein 4.2 and Rh-associated glycoprotein content in all patient RBCs, (2) increased endoplasmic reticulum remnants and endocytosis proteins in nonsplenectomized patients, and (3) increased lysosomal and mitochondrial remnants in splenectomized patients. Our study paves the way for a better understanding of the involvement of septins in RBC membrane biophysical properties. In addition, the lack of restoration of septin-independent cryohemolysis by splenectomy may call into question its recommendation in specific cases., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The endometrium plays a crucial role in reproduction and, in humans, is cyclically remodeled under hormonal control. Estradiol favors tissue proliferation whereas progesterone inhibits tissue growth and induces morphological changes. Endometriosis is often associated with fertility issues and with exacerbated estrogen and reduced progesterone concentration or response in the eutopic endometrium. However, underlying mechanisms remain unclear. Progesterone Receptor Membrane Component (PGRMC) 1 is a protein able to modulate progesterone response and its murine knockout reduced fertility. However, the precise spatiotemporal pattern of PGRMC1 expression in the human endometrium is still poorly characterized. We investigated variations of eutopic endometrial PGRMC1 expression by combining RT-qPCR, immunofluorescence and in situ hybridization. We found that PGRMC1 expression progressively increases during the proliferative phase and decreases during the secretory phase. However, immunolabeling and identification of mRNA-containing cells were regularly heterogeneous in samples, according to tissue depth, with a gradient extending from the surface epithelium towards the basalis. There was no significant difference in PGRMC1 mRNA amounts between patients with or without endometriosis or adenomyosis, for any phase of the menstrual cycle, but cells with strong or moderate PGRMC1 immunolabeling were reduced during the proliferative phase in endometriotic patients. In conclusion, although the cyclical variation of PGRMC1 expression globally follows fluctuation of ovarian steroids, further work is required to precisely characterize hormonal control and identify the additional levels of regulation responsible for local adjustment of PGRMC1 concentration. This is particularly important in the light of recent studies emphasizing the correlation between adequate PGRMC1 amounts and fertility., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Micro-RNAs (miRNAs) are small non-coding RNAs that regulate gene expression, mainly at mRNA post-transcriptional level. Functional maturation of most miRNAs requires processing of the primary transcript by Dicer, an RNaseIII-type enzyme. To date, the importance of miRNA function for normal organogenesis has been demonstrated in several mouse models of tissue-specific Dicer inactivation. However, the role of miRNAs in thyroid development has not yet been addressed. For the present study, we generated mouse models in which Dicer expression has been inactivated at two different stages of thyroid development in thyroid follicular cells. Regardless of the time of Dicer invalidation, the early stages of thyroid organogenesis, preceding folliculogenesis, were unaffected by the loss of small RNAs, with a bilobate gland in place. Nevertheless, Dicer mutant mice were severely hypothyroid and died soon after weaning unless they were substituted with T4. A conspicuous follicular disorganization was observed in Dicer mutant thyroids together with a strong down regulation of Nis expression. With increasing age, the thyroid tissue showed characteristics of neoplastic alterations as suggested by a marked proliferation of follicular cells and an ongoing de-differentiation in the center of the thyroid gland, with a loss of Pax8, FoxE1, Nis and Tpo expression. Together, our data show that loss of miRNA maturation due to Dicer inactivation severely disturbs functional thyroid differentiation. This suggests that miRNAs are mandatory to fine-tune the expression of thyroid specific genes and to maintain thyroid tissue homeostasis.

The effects of changes in macrophage iron status, induced by single or multiple iron injections, iron depletion or pregnancy, on both immune function and mRNA expression of genes involved in iron influx and egress have been evaluated. Macrophages isolated from iron deficient rats, or pregnant rats at day 21 of gestation, either supplemented with a single dose of iron dextran, 10 mg, at the commencement of pregnancy, or not, showed significant increases of macrophage ferroportin mRNA expression, which was paralleled by significant decreases in hepatic Hamp mRNA expression. IRP activity in macrophages was not significantly altered by iron status or the inducement of pregnancy +/- a single iron supplement. Macrophage immune function was significantly altered by iron supplementation and pregnancy. Iron supplementation, alone or combined with pregnancy, increased the activities of both NADPH oxidase and nuclear factor kappa B (NFkappaB). In contrast, the imposition of pregnancy reduced the ability of these parameters to respond to an inflammatory stimuli. Increasing iron status, if only marginally, will reduce the ability of macrophages to mount a sustained response to inflammation as well as altering iron homeostatic mechanisms.

Transcription factors of the ONECUT class, whose prototype is HNF-6, contain a single cut domain and a divergent homeodomain characterized by a phenylalanine at position 48 and a methionine at position 50. The cut domain is required for DNA binding. The homeodomain is required either for DNA binding or for transcriptional stimulation, depending on the target gene. Transcriptional stimulation by the homeodomain involves the F48M50 dyad. We investigate here how HNF-6 stimulates transcription. We identify transcriptionally active domains of HNF-6 that are conserved among members of the ONECUT class and show that the cut domain of HNF-6 participates to DNA binding and, via a LXXLL motif, to transcriptional stimulation. We also demonstrate that, on a target gene to which HNF-6 binds without requirement for the homeodomain, transcriptional stimulation involves an interaction of HNF-6 with the coactivator CREB-binding protein (CBP). This interaction depends both on the LXXLL motif of the cut domain and on the F48M50 dyad of the homeodomain. On a target gene for which the homeodomain is required for DNA binding, but not for transcriptional stimulation, HNF-6 interacts with the coactivator p300/CBP-associated factor but not with CBP. These data show that a transcription factor can act via different, sequence-specific, mechanisms that combine distinct modes of DNA binding with the use of different coactivators.

Smad4 is defined as the common-mediator Smad (co-Smad) required for transducing signals for all TGF-beta superfamily members. This paper describes two Smad4s in Xenopus: XSmad4alpha, which is probably the Xenopus orthologue of human Smad4, and a distinct family member, XSmad4beta, which differs primarily at the extreme N-terminus and in the linker region. Both XSmad4s act as co-Smads, forming ligand-dependent complexes with receptor-regulated Smads 1 and 2 and synergizing with them to activate transcription of mesodermal genes in Xenopus embryos. The two XSmad4 genes have reciprocal temporal expression patterns in Xenopus embryos and are expressed in varying ratios in adult tissues, suggesting distinct functional roles in vivo. XSmad4beta is the predominant maternal co-Smad and we go on to demonstrate its role in the transcriptional regulation of early mesodermal genes. We have identified two distinct nuclear complexes that bind the activin-responsive element of the Xenopus Mix.2 promoter: one formed in response to high levels of activin signaling and the other activated by endogenous signaling pathways. Using specific antisera we demonstrate the presence of endogenous XSmad4beta and also XSmad2 in both of these complexes, and our data indicate that the DNA-binding components of the complexes are different. Furthermore, we show that the presence of these complexes in the nucleus perfectly correlates with the transcriptional activity of the target gene, Mix.2, and we show that one of the XSmad4beta-containing transcription factor complexes undergoes a developmentally regulated nuclear translocation., (Copyright 1999 Academic Press.)

Glucocorticoids exert their effects on gene transcription through ubiquitous receptors that bind to regulatory sequences present in many genes. These glucocorticoid receptors are present in all cell types, yet glucocorticoid action is controlled in a tissue-specific way. One mechanism for this control relies on tissue-specific transcriptional activators that bind in the vicinity of the glucocorticoid receptor and are required for receptor action. We now describe a gene-specific and tissue-specific inhibitory mechanism through which glucocorticoid action is repressed by a tissue-restricted transcription factor, hepatocyte nuclear factor-6 (HNF-6). HNF-6 inhibits the glucocorticoid-induced stimulation of two genes coding for enzymes of liver glucose metabolism, namely 6-phosphofructo-2-kinase and phosphoenolpyruvate carboxykinase. Binding of HNF-6 to DNA is required for inhibition of glucocorticoid receptor activity. In vitro and in vivo experiments suggest that this inhibition is mediated by a direct HNF-6/glucocorticoid receptor interaction involving the amino-terminal domain of HNF-6 and the DNA-binding domain of the receptor. Thus, in addition to its known property of stimulating transcription of liver-expressed genes, HNF-6 can antagonize glucocorticoid-stimulated gene transcription.

Insulin can inhibit the stimulatory effect of glucocorticoid hormones on the transcription of genes coding for enzymes involved in glucose metabolism. We reported earlier that insulin inhibits the glucocorticoid-stimulated transcription of the gene coding for liver 6-phosphofructo-2-kinase (PFK-2). To elucidate the mechanism of these hormonal effects, we have studied the regulatory regions of the PFK-2 gene in transfection experiments. We found that both glucocorticoids and insulin act via the glucocorticoid response unit (GRU) located in the first intron. Footprinting experiments showed that the GRU binds not only the glucocorticoid receptor (GR), but also ubiquitous [nuclear factor I (NF-I)] and liver-enriched [hepatocyte nuclear factor (HNF)-3, HNF-6, CAAT/enhancer binding protein (C/EBP)] transcription factors. Site-directed mutational analysis of the GRU revealed that these factors modulate glucocorticoid action but that none of them seems to be individually involved in the inhibitory effect of insulin. We did not find an insulin response element in the GRU, but we showed that insulin targets the GR. Insulin-induced inhibition of the glucocorticoid stimulation required the ligand-binding domain of the GR. Finally, the insulin-signaling cascade involved was independent of the phosphatidylinositol-3-kinase and mitogen-activated protein kinase pathways. Together, these results suggest that insulin acts on the PFK-2 gene via another pathway and targets either the GR in its ligand-binding domain or a cofactor interacting with this domain.

Transcription from the liver promoter of a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) gene depends on the presence of glucocorticoids that act via a glucocorticoid response unit (GRU) located in the first intron. The promoter and the GRU are in a constitutively open chromatin configuration. To determine how glucocorticoids would affect factor binding to the GRU in absence of chromatin remodeling, we have used a combination of in vitro DNA-binding assays and in vivo genomic footprinting in rat hepatocytes and hepatoma cells. We found that, in the absence of glucocorticoids, the GRU binds nuclear factor-I (NF-I). Glucocorticoid treatment modified factor binding to the NF-I site and induced the binding of hepatocyte nuclear factor-3 (HNF-3). Transfection assays showed that HNF-3 cooperates with the glucocorticoid receptor in stimulating transcription. In contrast with the lack of effect of glucocorticoids on factor binding to constitutively open GRUs of other genes, HNF-3 binding to the open PFK-2 GRU was hormone-dependent. Therefore, the PFK-2 GRU behaves as a novel type of GRU.