MicroRNA-486-3p Regulates γ-Globin Expression in Human Erythroid Cells by Directly Modulating BCL11A

MicroRNAs (miRNAs) play key roles in modulating a variety of cellular processes through repression of mRNAs target. The functional relevance of microRNAs has been proven in normal and malignant hematopoiesis. While analyzing miRNAs expression profile in unilineage serum-free liquid suspension unilineage cultures of peripheral blood CD34⁺ hematopoietic progenitor cells (HPCs) through the erythroid, megakaryocytic, granulocytic and monocytic pathways, we identified miR-486-3p as mainly expressed within the erythroid lineage. We showed that miR-486-3p regulates BCL11A expression by binding to the extra-long isoform of BCL11A 3′UTR. Overexpression of miR-486-3p in erythroid cells resulted in reduced BCL11A protein levels, associated to increased expression of γ-globin gene, whereas inhibition of physiological miR-486-3p levels increased BCL11A and, consequently, reduced γ-globin expression. Thus, miR-486-3p regulating BCL11A expression might contributes to fetal hemoglobin (HbF) modulation and arise the question as to what extent this miRNA might contribute to different HbF levels observed among β-thalassemia patients. Erythroid cells, differentiated from PB CD34⁺ cells of a small cohort of patients affected by major or intermedia β-thalassemia, showed miR-486-3p levels significantly higher than those observed in normal counterpart. Importantly, in these patients, miR-486-3p expression correlates with increased HbF synthesis. Thus, our data indicate that miR-486-3p might contribute to different HbF levels observed among thalassemic patients and, possibly, to the clinical severity of the disease.     

MicroRNA-153 Physiologically Inhibits Expression of Amyloid-β Precursor Protein in Cultured Human Fetal Brain Cells and Is Dysregulated in a Subset of Alzheimer Disease Patients

Regulation of amyloid-β (Aβ) precursor protein (APP) expression is complex. MicroRNAs (miRNAs) are expected to participate in the molecular network that controls this process. The composition of this network is, however, still undefined. Elucidating the complement of miRNAs that regulate APP expression should reveal novel drug targets capable of modulating Aβ production in AD. Here, we investigated the contribution of miR-153 to this regulatory network. A miR-153 target site within the APP 3'-untranslated region (3'-UTR) was predicted by several bioinformatic algorithms. We found that miR-153 significantly reduced reporter expression when co-transfected with an APP 3'-UTR reporter construct. Mutation of the predicted miR-153 target site eliminated this reporter response. miR-153 delivery in both HeLa cells and primary human fetal brain cultures significantly reduced APP expression. Delivery of a miR-153 antisense inhibitor to human fetal brain cultures significantly elevated APP expression. miR-153 delivery also reduced expression of the APP paralog APLP2. High functional redundancy between APP and APLP2 suggests that miR-153 may target biological pathways in which they both function. Interestingly, in a subset of human AD brain specimens with moderate AD pathology, miR-153 levels were reduced. This same subset also exhibited elevated APP levels relative to control specimens. Therefore, endogenous miR-153 inhibits expression of APP in human neurons by specifically interacting with the APP 3'-UTR. This regulatory interaction may have relevance to AD etiology, where low miR-153 levels may drive increased APP expression in a subset of AD patients.     

Ceramide Stimulates ABCA12 Expression via Peroxisome Proliferator-activated Receptor �� in Human Keratinocytes

ABCA12 (ATP binding cassette transporter, family 12) is a cellular membrane transporter that facilitates the delivery of glucosylceramides to epidermal lamellar bodies in keratinocytes, a process that is critical for permeability barrier formation. Following secretion of lamellar bodies into the stratum corneum, glucosylceramides are metabolized to ceramides, which comprise ∼50% of the lipid in stratum corneum. Gene mutations of ABCA12 underlie harlequin ichthyosis, a devastating skin disorder characterized by abnormal lamellar bodies and a severe barrier abnormality. Recently we reported that peroxisome proliferator-activated receptor (PPAR) and liver X receptor activators increase ABCA12 expression in human keratinocytes. Here we demonstrate that ceramide (C₂-Cer and C₆-Cer), but not C₈-glucosylceramides, sphingosine, or ceramide 1-phosphate, increases ABCA12 mRNA expression in a dose- and time-dependent manner. Inhibitors of glucosylceramide synthase, sphingomyelin synthase, and ceramidase and small interfering RNA knockdown of human alkaline ceramidase, which all increase endogenous ceramide levels, also increased ABCA12 mRNA levels. Moreover, simultaneous treatment with C₆-Cer and each of these same inhibitors additively increased ABCA12 expression, indicating that ceramide is an important inducer of ABCA12 expression and that the conversion of ceramide to other sphingolipids or metabolites is not required. Finally, both exogenous and endogenous ceramides preferentially stimulate PPARδ expression (but not other PPARs or liver X receptors), whereas PPARδ knockdown by siRNA transfection specifically diminished the ceramide-induced increase in ABCA12 mRNA levels, indicating that PPARδ is a mediator of the ceramide effect. Together, these results show that ceramide, an important lipid component of epidermis, up-regulates ABCA12 expression via the PPARδ-mediated signaling pathway, providing a substrate-driven, feed-forward mechanism for regulating this key lipid transporter.The outermost layer of mammalian epidermis, the stratum corneum, is essential for permeability barrier function and critical for terrestrial life. The stratum corneum consists of terminally differentiated, anucleate keratinocytes, or corneocytes, surrounded by lipid-enriched lamellar membranes composed of three major lipids, ceramides, cholesterol, and free fatty acids (1). These lipids are delivered to the extracellular spaces of the stratum corneum through exocytosis of lamellar body contents from outermost stratum granulosum cells (2). Mature lamellar bodies contain primarily cholesterol, phospholipids, and glucosylceramides (3). Following lamellar body secretion, the secreted phospholipids and glucosylceramides are converted to free fatty acids and ceramides by phospholipases and β-glucocerebrosidase, respectively (1, 4). ABCA12 (ATP binding cassette transporter, family 12), a lipid transporter predominantly expressed in epidermis, has been shown to play a vital role in the formation of mature lamellar bodies (5, 6), although how this transporter is regulated remains unresolved.ABCA12 is a member of the ABCA subfamily of transporters, which are involved in the transport of a variety of lipids (7). Mutations in ABCA1 cause Tangier disease, which is due to a defect in transporting cholesterol and phospholipids from intracellular lipid stores to apolipoproteins, particularly apolipoprotein A-I (8–11). Mutations in ABCA3 cause neonatal respiratory failure due to a defect in surfactant transport from alveolar type II cells into the alveolar space (12). Mutations in ABCA4 cause Stargardt''s macular degeneration, with visual loss due to a defect in transporting phosphatidylethanolamine-retinylidene out of retinal pigment cells (13).Recently, mutations in ABCA12 have been shown to cause harlequin ichthyosis and a subgroup of lamellar ichthyosis, two disorders of keratinization (5, 14, 15). ABCA12 mutations lead to an abnormality in lamellar body formation, a decrease in lamellar membranes in the extracellular spaces of the stratum corneum, an accumulation of glucosylceramide in the epidermis with a reduction in ceramide (16), and ultimately loss of permeability barrier function (17), which in harlequin ichthyosis can result in neonatal lethality (5, 15). Strikingly, genetic correction of ABCA12 deficiency in patients'' keratinocytes by gene transfer normalized loading of glucosylceramides into lamellar bodies (5). These studies demonstrate a critical role for ABCA12 in epidermal physiology, specifically in the formation of mature lamellar bodies and subsequent permeability barrier homeostasis. Hence, it is crucial to understand how ABCA12 is regulated.Our laboratory recently demonstrated that activation of peroxisome proliferator-activated receptor (PPARδ and PPARγ) or liver X receptor (LXR) stimulates ABCA12 expression in cultured human keratinocytes (18). Both PPARs and LXR are important lipid sensors that stimulate keratinocyte differentiation and enhance permeability barrier function (19). Additionally, PPARα and -δ as well as LXR activators stimulate ceramide synthesis in keratinocytes (20, 21). Likewise, ceramide synthesis increases in keratinocytes during differentiation, foreshadowing the formation of lamellar bodies (22, 23).In addition to serving as structural membrane components, ceramides are also important signaling molecules that can induce growth arrest, differentiation, and apoptosis in various cells, including keratinocytes (24–26). Moreover, distal ceramide metabolites, sphingosine and sphingosine-1-phosphate (Fig. 1), are also important signaling molecules (27).Open in a separate windowFIGURE 1.The central role of ceramide in sphingolipid metabolism in keratinocytes. C1P, ceramide 1-phosphate; Sph, sphingosine; S1P, sphingosine-1-phosphate; GlcCer, glucosylceramide; SM, sphingomyelin.It is well established that the expression of ABCA1 is regulated by cellular cholesterol levels in many cell types, including keratinocytes (28). Cholesterol, if metabolized to certain oxysterols, can activate LXR, which then stimulates ABCA1 expression and the transport of cholesterol out of cells (29). This example of feed-forward regulation leads us to hypothesize that either ceramide or a metabolite of ceramide might stimulate ABCA12 expression, thereby leading to an increase in the transport of glucosylceramides into maturing lamellar bodies. Here, we provide evidence that ceramide stimulates ABCA12 expression in keratinocytes via a mechanism involving PPARδ signaling.     

Inflammation Anergy in Human Intestinal Macrophages Is Due to Smad-induced I��B�� Expression and NF-��B Inactivation

Human intestinal macrophages contribute to tissue homeostasis in noninflamed mucosa through profound down-regulation of pro-inflammatory cytokine release. Here, we show that this down-regulation extends to Toll-like receptor (TLR)-induced cytokine release, as intestinal macrophages expressed TLR3–TLR9 but did not release cytokines in response to TLR-specific ligands. Likely contributing to this unique functional profile, intestinal macrophages expressed markedly down-regulated adapter proteins MyD88 and Toll interleukin receptor 1 domain-containing adapter-inducing interferon β, which together mediate all TLR MyD88-dependent and -independent NF-κB signaling, did not phosphorylate NF-κB p65 or Smad-induced IκBα, and did not translocate NF-κB into the nucleus. Importantly, transforming growth factor-β released from intestinal extracellular matrix (stroma) induced identical down-regulation in the NF-κB signaling and function of blood monocytes, the exclusive source of intestinal macrophages. Our findings implicate stromal transforming growth factor-β-induced dysregulation of NF-κB proteins and Smad signaling in the differentiation of pro-inflammatory blood monocytes into noninflammatory intestinal macrophages.     

Glu-333 of Nicastrin Directly Participates in ��-Secretase Activity

γ-Secretase is a proteolytic membrane complex that processes a variety of substrates including the amyloid precursor protein and the Notch receptor. Earlier we showed that one of the components of this complex, nicastrin (NCT), functions as a receptor for γ-secretase substrates. A recent report challenged this, arguing instead that the Glu-333 residue of NCT predicted to participate in substrate recognition only participates in γ-secretase complex maturation and not in activity per se. Here, we present evidence that Glu-333 directly participates in γ-secretase activity. By normalizing to the active pool of γ-secretase with two separate methods, we establish that γ-secretase complexes containing NCT-E333A are indeed deficient in intrinsic activity. We also demonstrate that the NCT-E333A mutant is deficient in its binding to substrates. Moreover, we find that the cleavage of substrates by γ-secretase activity requires a free N-terminal amine but no minimal length of the extracellular N-terminal stub. Taken together, these studies provide further evidence supporting the role of NCT in substrate recognition. Finally, because γ-secretase cleaves itself during its maturation and because NCT-E333A also shows defects in γ-secretase complex maturation, we present a model whereby Glu-333 can serve a dual role via similar mechanisms in the recruitment of both Type 1 membrane proteins for activity and the presenilin intracellular loop during complex maturation.The brains of Alzheimer disease patients are characterized by dense neuritic plaques that consist of the insoluble β-amyloid peptide (Aβ)² and neurons containing neurofibrillary tangles of the Tau protein (1, 2). The Aβ peptide is produced via the sequential proteolysis of APP by β- and γ-secretase (3). γ-secretase is a multisubunit complex consisting of at least four proteins: presenilin (PS), NCT, APH-1, and PEN-2, all of which are necessary and sufficient for activity (4–9). The formation of the γ-secretase complex is tightly controlled, with an ordered assembly of subunits coupled to spatial restriction (10). It is believed that the last step of the complicated γ-secretase maturation and activation process involves in cis endoproteolysis of the PS holoprotein (11–13). It is this form of γ-secretase with PS in its N- and C-terminal fragments (NTF and CTF, respectively) that represents the fully mature, proteolytically active enzyme.γ-Secretase is a unique protease that cleaves within the lipid bilayer a large number of Type 1 single transmembrane-spanning proteins that vary widely in their sequence and size (14–16). In a previous report, we demonstrated that NCT functions as a substrate receptor for γ-secretase (4). In that report, we showed that NCT recruits substrates that have had their large extracellular domains first removed by an upstream protease in a process termed “ectodomain shedding.” This process generates a new, short extracellular stub with a free N terminus, which is required for proteolysis by γ-secretase. We also established that Glu-333 of NCT participates in activity within the larger context of the DYIGS and peptidase-like (DAP) domain, which shares distant homology to amino- and carboxypeptidases. A recent study by Chávez-Gutiérrez et al. (17) confirmed that mutations at the equivalent rodent residue impair γ-secretase. However, the authors attributed the reduction in activity to a role for Glu-333 in γ-secretase maturation but not directly in activity per se. Although a role for NCT and Glu-333 in γ-secretase assembly and maturation is consistent with our early work (4, 18, 19), the authors'' conclusion that mature γ-secretase complexes containing the Glu-333 mutant NCT are fully active presents a challenge to the model that NCT is a receptor for γ-secretase substrates in mature, active enzyme. Although PS-NTF or -CTF alone is an adequate measure of active γ-secretase complexes, Chávez-Gutiérrez et al. (17) measured specific activity by normalizing γ-secretase products to the sum of PS1-CTF and PEN-2 presumably due to the levels of PS-NTF/CTF by themselves being at the detection limit of Western blotting with electrochemiluminescence (ECL). Such an approach has caveats, as normalizing to the sum of PS1 and PEN-2 does not represent a measurement of the intrinsic activity per single, active enzyme; rather, this mode of normalization instead skews the data to minimize the effects of the mutations, especially when compounded with the unreliability of ECL measurement at the detection limit of Western blotting. Indeed, normalizing to the amount of mature, active γ-secretase in a rigorous, quantitative manner would be necessary to accurately compare the intrinsic activities of wild-type and mutant enzymes.In this study we used two γ-secretase reconstitution methods, including one that bypasses endoproteolysis and two separate normalization approaches to demonstrate that γ-secretase complexes containing NCT-E333A are indeed intrinsically less active than wild-type NCT. We show that this mutant is deficient in its ability to directly bind to γ-secretase substrates. Moreover, we confirm our observations with a second γ-secretase substrate, C83, which is itself the physiological product of α-secretase cleavage of APP. We also examine a series of substrate truncation mutants and find that γ-secretase can cleave substrates that lack the entire extracellular domain, provided that such substrates also contain a free N-terminal amine. Taken together, we conclude that Glu-333 participates directly in activity after γ-secretase complex maturation. Finally, we put forth a model wherein the dual role of Glu-333 in γ-secretase maturation and substrate recognition could be explained in the context of NCT being a substrate receptor. In this model Glu-333 partakes in the recruitment of not only the ectodomain-shed Type 1 membrane proteins but also of the intracellular loop of PS for its endoproteolysis, a hallmark event of γ-secretase maturation and activation.     

High-Glucose Environment Inhibits p38MAPK Signaling and Reduces Human β-Defensin-3 Expression [corrected] in Keratinocytes

Diabetes mellitus is characterized by elevated plasma glucose and increased rates of skin infections. Altered immune responses have been suggested to contribute to this prevalent complication, which involves microbial invasion. In this study we explored the effects of a high-glucose environment on the innate immunity of keratinocytes by focusing on β defensin-3 (BD3) using in vivo and in vitro models. Our results demonstrated that the perilesional skins of diabetic rats failed to show enhanced BD3 expression after wounding. In addition, high-glucose treatment reduced human BD3 (hBD3) expression of cultured human keratinocytes. This pathogenic process involved inhibition of p38MAPK signaling, an event that resulted from increased formation of advanced glycation end products. On the other hand, toll-like receptor-2 expression and function of cultured keratinocytes were not significantly affected by high-glucose treatment. In summary, high-glucose conditions inhibited the BD3 expression of epidermal keratinocytes, which in turn contributed to the frequent occurrences of infection associated with diabetic wounding.