A CD44/Brg1 nuclear complex confers mesenchymal progenitor cells with enhanced fibrogenicity in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease. We previously identified fibrogenic mesenchymal progenitor cells (MPCs) in the lungs of patients with IPF who serve as drivers of progressive fibrosis. Recent single-cell RNA sequencing work revealed that IPF MPCs with the highest transcriptomic network entropy differ the most from control MPCs and that increased CD44 was a marker of these IPF MPCs. We hypothesize that IPF MPCs with high CD44 (CD44hi) expression will display enhanced fibrogenicity. We demonstrate that CD44-expressing MPCs are present at the periphery of the IPF fibroblastic focus, placing them in regions of active fibrogenesis. In a humanized mouse xenograft model, CD44hi IPF MPCs are more fibrogenic than CD44lo IPF MPCs, and knockdown of CD44 diminishes their fibrogenicity. CD44hi IPF MPCs display increased expression of pluripotency markers and enhanced self-renewal compared with CD44lo IPF MPCs, properties potentiated by IL-8. The mechanism involves the accumulation of CD44 within the nucleus, where it associates with the chromatin modulator protein Brahma-related gene 1 (Brg1) and the zinc finger E-box binding homeobox 1 (Zeb1) transcription factor. This CD44/Brg1/Zeb1 nuclear protein complex targets the Sox2 gene, promoting its upregulation and self-renewal. Our data implicate CD44 interaction with the epigenetic modulator protein Brg1 in conveying IPF MPCs with cell-autonomous fibrogenicity.

Lumacaftor/ivacaftor therapy fails to increase insulin secretion in F508del/F508del CF patients.

BACKGROUND: Glucose tolerance abnormalities including cystic fibrosis related diabetes (CFRD) are common in patients with cystic fibrosis (CF). The underlying pathophysiology is not fully understood. Emerging evidence suggests that CFTR dysfunction may directly or indirectly impact ß-cell function, offering the potential for improvement with CFTR modulator therapy. In small pilot studies, treatment with ivacaftor improved insulin secretion in patients with the G551D CFTR mutation. In the current study, we examined the impact of lumacaftor/ivacaftor therapy on glucose tolerance and insulin secretion in patients with CF who were homozygous for the F508del mutation. METHODS: 39 subjects from the PROSPECT Part B study who had been prescribed lumacaftor/ivacaftor by their CF care team at a CF Foundation's Therapeutic Development Network center were recruited. Subjects underwent 2-hour oral glucose tolerance tests (OGTTs) at baseline prior to first dose of lumacaftor/ivacaftor, and at 3, 6 and 12 months on therapy. OGTT glucose, insulin and c-peptide parameters were compared. RESULTS: Compared to baseline, OGTT fasting and 2 hour glucose levels, glucose area under the curve, insulin area under the curve and time to peak insulin level were not significantly different at 3, 6 and 12 months on lumacaftor/ivacaftor therapy. Similarly, C-peptide levels were no different. CONCLUSIONS: Lumacaftor/ivacaftor therapy did not improve insulin secretion or glucose tolerance in patients with CF who were homozygous for the F508del mutation.

Single-cell RNA sequencing reveals that lung mesenchymal progenitor cells in IPF exhibit pathological features early in their differentiation trajectory.

In Idiopathic Pulmonary Fibrosis (IPF), there is unrelenting scarring of the lung mediated by pathological mesenchymal progenitor cells (MPCs) that manifest autonomous fibrogenicity in xenograft models. To determine where along their differentiation trajectory IPF MPCs acquire fibrogenic properties, we analyzed the transcriptome of 335 MPCs isolated from the lungs of 3 control and 3 IPF patients at the single-cell level. Using transcriptional entropy as a metric for differentiated state, we found that the least differentiated IPF MPCs displayed the largest differences in their transcriptional profile compared to control MPCs. To validate entropy as a surrogate for differentiated state functionally, we identified increased CD44 as a characteristic of the most entropic IPF MPCs. Using FACS to stratify IPF MPCs based on CD44 expression, we determined that CD44hi IPF MPCs manifested an increased capacity for anchorage-independent colony formation compared to CD44lo IPF MPCs. To validate our analysis morphologically, we used two differentially expressed genes distinguishing IPF MPCs from control (CD44, cell surface; and MARCKS, intracellular). In IPF lung tissue, pathological MPCs resided in the highly cellular perimeter region of the fibroblastic focus. Our data support the concept that IPF fibroblasts acquire a cell-autonomous pathological phenotype early in their differentiation trajectory.

Dicer1 Deficiency in the Idiopathic Pulmonary Fibrosis Fibroblastic Focus Promotes Fibrosis by Suppressing MicroRNA Biogenesis.

RATIONALE: The lung extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) mediates progression of fibrosis by decreasing fibroblast expression of miR-29 (microRNA-29), a master negative regulator of ECM production. The molecular mechanism is undefined. IPF-ECM is stiffer than normal. Stiffness drives fibroblast ECM production in a YAP (yes-associated protein)-dependent manner, and YAP is a known regulator of miR-29. Therefore, we tested the hypothesis that negative regulation of miR-29 by IPF-ECM was mediated by mechanotransduction of stiffness. OBJECTIVES: To determine how IPF-ECM negatively regulates miR-29. METHODS: We decellularized lung ECM using detergents and prepared polyacrylamide hydrogels of defined stiffness by varying acrylamide concentrations. Mechanistic studies were guided by immunohistochemistry of IPF lung and used cell culture, RNA-binding protein assays, and xenograft models. MEASUREMENTS AND MAIN RESULTS: Contrary to our hypothesis, we excluded fibroblast mechanotransduction of ECM stiffness as the primary mechanism deregulating miR-29. Instead, systematic examination of miR-29 biogenesis revealed a microRNA processing defect that impeded processing of miR-29 into its mature bioactive forms. Immunohistochemical analysis of the microRNA processing machinery in IPF lung specimens revealed decreased Dicer1 expression in the procollagen-rich myofibroblastic core of fibroblastic foci compared with the focus perimeter and adjacent alveolar walls. Mechanistically, IPF-ECM increased association of the Dicer1 transcript with RNA binding protein AUF1 (AU-binding factor 1), and Dicer1 knockdown conferred primary human lung fibroblasts with cell-autonomous fibrogenicity in zebrafish and mouse lung xenograft models. CONCLUSIONS: Our data identify suppression of fibroblast Dicer1 expression in the myofibroblast-rich IPF fibroblastic focus core as a central step in the mechanism by which the ECM sustains fibrosis progression in IPF.

The hepatitis C viral nonstructural protein 5A stabilizes growth-regulatory human transcripts.

Numerous mammalian proto-oncogene and other growth-regulatory transcripts are upregulated in malignancy due to abnormal mRNA stabilization. In hepatoma cells expressing a hepatitis C virus (HCV) subgenomic replicon, we found that the viral nonstructural protein 5A (NS5A), a protein known to bind to viral RNA, also bound specifically to human cellular transcripts that encode regulators of cell growth and apoptosis, and this binding correlated with transcript stabilization. An important subset of human NS5A-target transcripts contained GU-rich elements, sequences known to destabilize mRNA. We found that NS5A bound to GU-rich elements in vitro and in cells. Mutation of the NS5A zinc finger abrogated its GU-rich element-binding and mRNA stabilizing activities. Overall, we identified a molecular mechanism whereby HCV manipulates host gene expression by stabilizing host transcripts in a manner that would promote growth and prevent death of virus-infected cells, allowing the virus to establish chronic infection and lead to the development of hepatocellular carcinoma.

Mesenchymal stem cells in the pathogenesis and treatment of bronchopulmonary dysplasia: a clinical review.

Advances in neonatal medicine have led to increased survival of infants born at the limits of viability, resulting in an increased incidence of bronchopulmonary dysplasia (BPD). BPD is a chronic lung disease of premature infants characterized by the arrest of alveolarization, fibroblast activation, and inflammation. BPD leads to significant morbidity and mortality in the neonatal period and is one of the leading causes of chronic lung disease in children. The past decade has brought a surge of trials investigating cellular therapies for the treatment of pulmonary diseases. Mesenchymal stem cells (MSCs) are of particular interest because of their ease of isolation, low immunogenicity, and anti-inflammatory and reparative properties. Clinical trials of MSCs have demonstrated short-term safety and tolerability; however, studies have also shown populations of MSCs with adverse pro-inflammatory and myofibroblastic characteristics. Cell-based therapies may represent the next breakthrough therapy for the treatment of BPD, however, there remain barriers to implementation as well as gaps in knowledge of the role of endogenous MSCs in the pathogenesis of BPD. Concurrent high-quality basic science, translational, and clinical studies investigating the fundamental pathophysiology underlying BPD, therapeutic mechanisms of exogenous MSCs, and logistics of translating cellular therapies will be important areas of future research.

Sporadic Insulinoma Presenting as Early Morning Night Terrors.

A 16-year-old boy with a recent diagnosis of night terrors was evaluated for recurrent early morning hypoglycemia after an early morning seizure. Evaluation in clinic with critical laboratories identified hyperinsulinemic hypoglycemia. Additional investigation revealed a sporadic insulinoma as the etiology of his hypoglycemia and all symptoms were resolved after pancreaticoduodenectomy. The importance of obtaining critical laboratory samples is highlighted and appropriate radiologic, medical, and pathologic testing is discussed. We additionally review the medical and surgical management of hyperinsulinemic hypoglycemia. A discussion of multiple endocrine neoplasia type 1 associated insulinomas is included as well. This case highlights the importance of considering hypoglycemia in the evaluation of night terrors and new-onset seizures.

T cell receptor cross-reactivity between similar foreign and self peptides influences naive cell population size and autoimmunity.

T cell receptor (TCR) cross-reactivity between major histocompatibility complex II (MHCII)-binding self and foreign peptides could influence the naive CD4(+) T cell repertoire and autoimmunity. We found that nonamer peptides that bind to the same MHCII molecule only need to share five amino acids to cross-react on the same TCR. This property was biologically relevant because systemic expression of a self peptide reduced the size of a naive cell population specific for a related foreign peptide by deletion of cells with cross-reactive TCRs. Reciprocally, an incompletely deleted naive T cell population specific for a tissue-restricted self peptide could be triggered by related microbial peptides to cause autoimmunity. Thus, TCR cross-reactivity between similar self and foreign peptides can reduce the size of certain foreign peptide-specific T cell populations and might allow T cell populations specific for tissue-restricted self peptides to cause autoimmunity after infection.

Alternative polyadenylation regulates CELF1/CUGBP1 target transcripts following T cell activation.

Alternative polyadenylation (APA) is an evolutionarily conserved mechanism for regulating gene expression. Transcript 3' end shortening through changes in polyadenylation site usage occurs following T cell activation, but the consequences of APA on gene expression are poorly understood. We previously showed that GU-rich elements (GREs) found in the 3' untranslated regions of select transcripts mediate rapid mRNA decay by recruiting the protein CELF1/CUGBP1. Using a global RNA sequencing approach, we found that a network of CELF1 target transcripts involved in cell division underwent preferential 3' end shortening via APA following T cell activation, resulting in decreased inclusion of CELF1 binding sites and increased transcript expression. We present a model whereby CELF1 regulates APA site selection following T cell activation through reversible binding to nearby GRE sequences. These findings provide insight into the role of APA in controlling cellular proliferation during biological processes such as development, oncogenesis and T cell activation.

Perspectives on the ARE as it turns 25 years old.

The AU-rich element (ARE) was discovered in 1986 as a conserved mRNA sequence found in the 3' untranslated region of the TNF-α transcript and other transcripts encoding cytokines and inflammatory mediators. Shortly thereafter, the ARE was shown to function as a regulator of mRNA degradation, and AREs were later shown to regulate other posttranscriptional mechanisms such as translation and mRNA localization. AREs coordinately regulate networks of chemokine, cytokine, and growth regulatory transcripts involved in cellular activation, proliferation, and inflammation. ARE-mediated regulation is carried out by a host of ARE-binding proteins, whose activity is regulated in a cell type and activation-dependent manner. The last 25 years of ARE research has offered insight into the mechanisms and regulation of ARE-mediated mRNA decay, and has provided a road map for the discovery of additional mRNA regulatory motifs. The future of ARE research will transition from a discovery phase to a phase focused on translating basic biological findings into novel therapeutic targets. Our understanding of ARE-mediated gene regulation and posttranscriptional control has implications for many fields of study including developmental biology, neuroscience, immunobiology, and cancer biology.

Regulation of CUG-binding protein 1 (CUGBP1) binding to target transcripts upon T cell activation.

The RNA-binding protein, CUG-binding protein 1 (CUGBP1), regulates gene expression at the levels of alternative splicing, mRNA degradation, and translation. We used RNA immunoprecipitation followed by microarray analysis to identify the cytoplasmic mRNA targets of CUGBP1 in resting and activated primary human T cells and found that CUGBP1 targets were highly enriched for the presence of GU-rich elements (GREs) in their 3'-untranslated regions. The number of CUGBP1 target transcripts decreased dramatically following T cell activation as a result of activation-dependent phosphorylation of CUGBP1 and decreased ability of CUGBP1 to bind to GRE-containing RNA. A large percentage of CUGBP1 target transcripts exhibited rapid and transient up-regulation, and a smaller percentage exhibited transient down-regulation following T cell activation. Many of the transiently up-regulated CUGBP1 target transcripts encode important regulatory proteins necessary for transition from a quiescent state to a state of cellular activation and proliferation. Overall, our results show that CUGBP1 binding to certain GRE-containing target transcripts decreased following T cell activation through activation-dependent phosphorylation of CUGBP1.

Global assessment of GU-rich regulatory content and function in the human transcriptome.

Unlike AU-rich elements (AREs) that are largely present in the 3'UTRs of many unstable mammalian mRNAs, the function and abundance of GU-rich elements (GREs) are poorly understood. We performed a genome-wide analysis and found that at least 5% of human genes contain GREs in their 3'UTRs with functional over-representation in genes involved in transcription, nucleic acid metabolism, developmental processes, and neurogenesis. GREs have similar sequence clustering patterns with AREs such as overlapping GUUUG pentamers and enrichment in 3'UTRs. Functional analysis using T-cell mRNA expression microarray data confirms correlation with mRNA destabilization. Reporter assays show that compared to AREs the ability of GREs to destabilize mRNA is modest and does not increase with the increasing number of overlapping pentamers. Naturally occurring GREs within U-rich contexts were more potent in destabilizing GFP reporter mRNAs than synthetic GREs with perfectly overlapping pentamers. Overall, we find that GREs bear a resemblance to AREs in sequence patterns but they regulate a different repertoire of genes and have different dynamics of mRNA decay. A dedicated resource on all GRE-containing genes of the human, mouse and rat genomes can be found at brp.kfshrc.edu.sa/GredOrg.

Analysis of CUGBP1 targets identifies GU-repeat sequences that mediate rapid mRNA decay.

CUG-repeat binding protein 1 (CUGBP1) mediates selective mRNA decay by binding to GU-rich elements (GREs) containing the sequence UGUUUGUUUGU found in the 3' untranslated region (UTR) of short-lived transcripts. We used an anti-CUGBP1 antibody to immunoprecipitate CUGBP1 from HeLa cytoplasmic extracts and analyzed the associated transcripts using oligonucleotide microarrays. We identified 613 putative mRNA targets of CUGBP1 and found that the UGUUUGUUUGU GRE sequence and a GU-repeat sequence were both highly enriched in the 3' UTRs of these targets. We showed that CUGBP1 bound specifically to the GU-repeat sequence and that insertion of this sequence into the 3' UTR of a beta-globin reporter transcript conferred instability to the transcript. Based on these results, we redefined the GRE to include this GU-repeat sequence. Our results suggest that CUGBP1 coordinately regulates the mRNA decay of a network of transcripts involved in cell growth, cell motility, and apoptosis.