BACKGROUND AND PURPOSE:Asthma is characterized by airway hyperresponsiveness (AHR), allergic inflammation, and airway remodelling. Although recent studies have shown that asthma pathophysiology involves P2X4 receptor activation, a potential link with chronic asthma remains to be explored. We investigated the effect of a novel P2X4 receptor antagonist BR11595 on allergen-induced airway responses in a guinea pig model of chronic asthma. EXPERIMENTAL APPROACH:Sensitized guinea pigs were exposed to saline or ovalbumin (OVA) once weekly via aerosolization for 12 weeks. BR11595 (10 mg·kg-1) was injected intraperitoneally five times per week, for four different regimens: all 12 weeks, first 6 weeks, last 6 weeks, or last week only. Airway responsiveness to histamine was assessed 24 h before and 6 h after OVA exposure in weeks 1, 6, and 12. Lung tissue inflammation and remodelling were determined 24 h after the last OVA exposure. KEY RESULTS:OVA induced AHR at weeks 1, 6, and 12 compared with saline-challenged animals. The AHR was less pronounced in week 12 compared with week 1. BR11595 significantly reduced OVA-induced AHR in week 6 in guinea pigs treated with BR11595 for 6 weeks. AHR in week 12 was reduced after BR11595 treatment in week 12 only, next to OVA-induced eosinophilia and Goblet cell hyperplasia, indicating an acute role of P2X4 receptors on chronic inflammation. CONCLUSION AND IMPLICATIONS:The P2X4-receptor antagonist BR11595 acutely inhibits AHR, eosinophilia, and Goblet cell hyperplasia after 12 weeks, indicating its potential as a therapeutic target for acute intervention of chronic asthma attacks or exacerbations.

Idiopathic pulmonary fibrosis (IPF) is a lethal disease with substantial unmet medical needs. While aberrant epithelial remodeling is a key factor in IPF progression, the molecular mechanisms behind this process remain elusive. Harnessing a 3D patient-derived organoid model and multi-omics approach, the first inventory of the connection between metabolic alteration, chromatin accessibility, and transcriptional regulation in IPF aberrant epithelial remodeling is provided. This remodeling is characterized by an increase in chromatin accessibility, particularly at JUNB motif-enriched promoter regions proximal to transcription start sites of metabolic and pro-fibrotic genes. Mechanistically, JUNB undergoes O-linked β-N-acetylglucosamine modification (O-GlcNAcylation), a critical step in modulating pro-fibrotic responses to chronic injury. This modification is pivotal in fostering the emergence of aberrant epithelial basal cells in the alveolar niche, a proposed driver of IPF pathology. Specific deletion of O-GlcNAcylation sites on JUNB attenuates the metaplastic differentiation of basal cells, thereby aiding in the restoration of the alveolar lineage. Together, the findings reveal a novel link between metabolic dysregulation and cell fate regulation at the chromatin level in fibrosis, mediated by the O-GlcNAc-JUNB axis, suggesting avenues for the development of new therapeutic strategies in IPF.

Patients with pulmonary fibrosis (PF) often experience exacerbations of their disease, characterised by a rapid, severe deterioration in lung function that is associated with high mortality. Whilst the pathobiology of such exacerbations is poorly understood, virus infection is a trigger. The present study investigated virus-induced injury responses of alveolar and bronchial epithelial cells (AECs and BECs, respectively) from patients with PF and age-matched controls (Ctrls). Air–liquid interface (ALI) cultures of AECs, comprising type I and II pneumocytes or BECs were inoculated with influenza A virus (H1N1) at 0.1 multiplicity of infection (MOI). Levels of interleukin-6 (IL-6), IL-36γ and IL-1β were elevated in cultures of AECs from PF patients (PF-AECs, n = 8–11), being markedly higher than Ctrl-AECs (n = 5–6), 48 h post inoculation (pi) (P<0.05); despite no difference in H1N1 RNA copy numbers 24 h pi. Furthermore, the virus-induced inflammatory responses of PF-AECs were greater than BECs (from either PF patients or controls), even though viral loads in the BECs were overall 2- to 3-fold higher than AECs. Baseline levels of the senescence and DNA damage markers, nuclear p21, p16 and H2AXγ were also significantly higher in PF-AECs than Ctrl-AECs and further elevated post-infection. Senescence induction using etoposide augmented virus-induced injuries in AECs (but not viral load), whereas selected senotherapeutics (rapamycin and mitoTEMPO) were protective. The present study provides evidence that senescence increases the susceptibility of AECs from PF patients to severe virus-induced injury and suggests targeting senescence may provide an alternative option to prevent or treat the exacerbations that worsen the underlying disease.

Dysfunctional interactions between fibroblasts and epithelial cells contribute to the progression of chronic lung diseases, including idiopathic pulmonary fibrosis (IPF). In this study, we utilized a coculture model of human small airway epithelial cells and fibroblasts to investigate intercellular communication during disease progression. Our transcriptomic and proteomic profiling reveal that fibroblasts repair epithelial cells in acute injury by boosting epithelial fatty acid metabolism; conversely, they exacerbate epithelial damage in chronic injury scenarios. By delineating regulators involved in these responses, we identified pentraxin 3 (PTX3) as a key antifibrotic factor secreted by fibroblasts in response to acute epithelial injury. Importantly, PTX3 levels are decreased in bronchoalveolar lavage (BAL) samples from IPF patients compared to non-fibrotic controls, indicating a potential link between diminished PTX3 levels and fibrosis progression. Furthermore, adding PTX3 to chronically injured epithelial-fibroblast cocultures mitigated the pro-fibrotic response and restored the epithelial barrier integrity. These findings highlight the dual roles of fibroblasts and the critical function of PTX3 in lung injury and repair, offering insights for therapeutic strategies. ### Competing Interest Statement All authors were employed by Boehringer Ingelheim Pharma GmbH & Co KG or by C.H. Boehringer Sohn AG and Co KG. This study was funded by Boehringer Ingelheim Pharma GmbH & Co KG.

Increased rate of aerobic glycolysis in idiopathic pulmonary fibrosis (IPF) is associated with pathological cellular phenotypes like epithelial barrier dysfunction, pro-fibrotic secretions and myofibroblast activation. Expression of lactate dehydrogenase (LDH), an enzyme involved in the final step of glycolysis and responsible for interconversion of pyruvate and lactate, is increased in IPF and the mouse bleomycin model lung tissue. Importantly, it has been shown that inhibition of glycolysis in bleomycin-induced lung fibrosis in mice led to a decreased collagen deposition and overall improve of the architecture of the lung. Here we investigate the potential of LDH inhibition in attenuating fibrosis-related features in human systems. To this end we pre-treated human small airway epithelial cells (SAEC) in air-liquid-interface setting with an LDH inhibitor and exposed them to TNFα to induce injury. LDH inhibition reduced the increased epithelial permeability, a hallmark of chronic lung diseases, in a dose-dependent manner. We further assessed the impact of prophylactic LDH inhibition on fibroblasts activation in a scar-in-a-jar model using primary material. Inhibition of LDH decreased Collagen I deposition and fiber formation in a dose-dependent manner. Additionally, inhibition of LDH in human monocyte-derived macrophages reduced the secretion of MMP9, a metalloprotease elevated in IPF and associated with aberrant lung remodeling, upon inflammatory and pro-fibrotic stimuli. Collectively, we show that inhibiting LDH and reducing lactate acidosis attenuates fibrosis-related features. Whether targeting LDH represents a new therapeutic concept for IPF will require assessment in in vivo preclinical models but recent data already provide a link between LDH5 and human fibrotic diseases.

Airway mucociliary regeneration and function are key players for airway defense and are impaired in chronic obstructive pulmonary disease (COPD). Using transcriptome analysis in COPD-derived bronchial biopsies, we observed a positive correlation between cilia-related genes and microRNA-449 (miR449). In vitro, miR449 was strongly increased during airway epithelial mucociliary differentiation. In vivo, miR449 was upregulated during recovery from chemical or infective insults. miR0449−/− mice (both alleles are deleted) showed impaired ciliated epithelial regeneration after naphthalene and Haemophilus influenzae exposure, accompanied by more intense inflammation and emphysematous manifestations of COPD. The latter occurred spontaneously in aged miR449−/− mice. We identified Aurora kinase A and its effector target HDAC6 as key mediators in miR449-regulated ciliary homeostasis and epithelial regeneration. Aurora kinase A is downregulated upon miR449 overexpression in vitro and upregulated in miR449−/− mouse lungs. Accordingly, imaging studies showed profoundly altered cilia length and morphology accompanied by reduced mucociliary clearance. Pharmacological inhibition of HDAC6 rescued cilia length and coverage in miR449−/− cells, consistent with its tubulin-deacetylating function. Altogether, our study establishes a link between miR449, ciliary dysfunction, and COPD pathogenesis.

Synchrotron-based imaging allows for detection of bronchiectasis-like phenotypes in mice with mucociliary clearance disorders https://bit.ly/3gXGdP3.

GPR4 is activated by acidosis in inflammation and hypoxia. COPD and bronchiectasis patients have reduced exhaled breath pH values around pH 7.1 suggesting disease-relevance of acidosis. GPR4 is highly expressed in lungs, where it regulates vascular permeability, leukocyte infiltration, and inflammatory response. GPR4 inhibition has thus been proposed to rescue COVID19 features incl. metabolic and respiratory acidosis, hyperinflammation, vessel leakage and edema formation. Additionally, GPR4 KO lowers blood pressure and enhances insulin sensitivity, both beneficial to COVID19 high-risk population. Here we show that GPR4 antagonism rescues edema, cytokine storm and leukocyte recruitment in experimental COPD-exacerbation and ARDS models. Additionally, it reduces kidney damage in an ischemic acute kidney injury model. Cellularly, GPR4 is highly expressed in activated Goblet cells of chronic lung disease patients and induced by IL1b and IL13 in human organotypic cultures. Its inhibition reduces inflammatory burden incl. mucus production. GPR4 is expressed in endothelia of diseased lungs, where it contributes to leukocyte recruitment, and in primary lung macrophages, where its inhibition reduces inflammatory response in a dose- and pH-dependent manner. Finally, RNA from human nasal swabs for Sars-Cov-2 diagnosis reveal correlation between GPR4 expression at test time and severity of COVID-19 severity at later stage. Altogether our data suggest that GPR4 antagonists may serve to reduce injury severity in lungs and kidneys of COVID19 patients. Additionally, early correlation between GPR4 expression in nasal swabs and future disease severity suggest potential as early predictive outcome biomarker.

The Histone Deacetylase 6 (HDAC6) enzyme has two deacetylase domains and is found almost exclusively in the cytoplasm. It deacetylates targets such as tubulin, cortactin, HSP90, KU70 and potentially the Treg-specific transcription factor FOXP3 but not histones under physiological conditions. COPD frequent exacerbators (COPD-FE) data suggest increased activity of HDAC6 and preclinical literature suggests HDAC6 inhibition/depletion rescues cilia shortening, mucociliary clearance defects and pulmonary barrier integrity, thus reducing susceptibility to acute lung injury. Additional literature describes functions in PAH, CF and IPF as well as non-pulmonary indications, with protection from various inflammatory diseases. Here we present the characterization of HDAC6 inhibitors in primary human bronchial epithelial cells grown at the air-liquid interface and Hdac6 KO mice in two preclinical models of COPD (elastase/LPS and cigarette smoke). HDAC6 inhibition and deficiency reduce mucus production and persistence of inflammation both in mice and in 3D human respiratory epithelia, with strong evidence for reducing MUC5AC positive cells as well as Mmp9 expression and neutrophilic inflammation. Thus, HDAC6 represents a solid pharmacological target with the potential to benefit COPD-FE patients, especially if suffering from chronic mucus hypersecretion. Method: H. Meier, F. Kölling, J. Auburger, L. Schneider, C. Noack, L. Dietz, D. Urrego, S. Schröder, I. Hagelschuer, T. Krämer

Rationale: Promoting endogenous pulmonary regeneration is crucial after damage to restore normal lungs and prevent the onset of chronic adult lung diseases.Objectives: To investigate whether the cell-cycle inhibitor p16INK4a limits lung regeneration after newborn bronchopulmonary dysplasia (BPD), a condition characterized by the arrest of alveolar development, leading to adult sequelae.Methods: We exposed p16INK4a-/- and p16INK4aATTAC (apoptosis through targeted activation of caspase 8) transgenic mice to postnatal hyperoxia, followed by pneumonectomy of the p16INK4a-/- mice. We measured p16INK4a in blood mononuclear cells of preterm newborns, 7- to 15-year-old survivors of BPD, and the lungs of patients with BPD.Measurements and Main Results: p16INK4a concentrations increased in lung fibroblasts after hyperoxia-induced BPD in mice and persisted into adulthood. p16INK4a deficiency did not protect against hyperoxic lesions in newborn pups but promoted restoration of the lung architecture by adulthood. Curative clearance of p16INK4a-positive cells once hyperoxic lung lesions were established restored normal lungs by adulthood. p16INK4a deficiency increased neutral lipid synthesis and promoted lipofibroblast and alveolar type 2 (AT2) cell development within the stem-cell niche. Besides, lipofibroblasts support self-renewal of AT2 cells into alveolospheres. Induction with a PPARγ (peroxisome proliferator-activated receptor γ) agonist after hyperoxia also increased lipofibroblast and AT2 cell numbers and restored alveolar architecture in hyperoxia-exposed mice. After pneumonectomy, p16INK4a deficiency again led to an increase in lipofibroblast and AT2 cell numbers in the contralateral lung. Finally, we observed p16INK4a mRNA overexpression in the blood and lungs of preterm newborns, which persisted in the blood of older survivors of BPD.Conclusions: These data demonstrate the potential of targeting p16INK4a and promoting lipofibroblast development to stimulate alveolar regeneration from childhood to adulthood.

Motile cilia serve vital functions in development, homeostasis and regeneration. We recently demonstrated that TAp73 is an essential transcriptional regulator of respiratory multiciliogenesis. Here, we show that TAp73 is expressed in multiciliated cells (MCCs) of diverse tissues. Analysis of TAp73 mutant animals revealed that TAp73 regulates Foxj1, Rfx2, Rfx3 , axonemal dyneins Dnali1 and Dnai1 , plays a pivotal role in the generation of MCCs in male and female reproductive ducts, and contributes to fertility. However, the function of MCCs in the brain appears to be preserved despite the loss of TAp73 , and robust activity of cilia-related networks is maintained in the absence of TAp73 . Notably, TAp73 loss leads to distinct changes in ciliogenic microRNAs: miR34bc expression is reduced, whereas the miR449 cluster is induced in diverse multiciliated epithelia. Among different MCCs, choroid plexus (CP) epithelial cells in the brain display prominent miR449 expression, whereas brain ventricles exhibit significant increase in miR449 levels along with an increase in the activity of ciliogenic E2F4/MCIDAS circuit in TAp73 mutant animals. Conversely, E2F4 induces robust transcriptional response from miR449 genomic regions. To address whether increased miR449 levels in the brain maintain the multiciliogenesis program in the absence of TAp73 , we deleted both TAp73 and miR449 in mice. Although loss of miR449 alone led to a mild ciliary defect in the CP, more pronounced ciliary defects and hydrocephalus were observed in the brain lacking both TAp73 and miR449 . In contrast, miR449 loss in other MCCs failed to enhance ciliary defects associated with TAp73 loss. Together, our study shows that, in addition to the airways, TAp73 is essential for generation of MCCs in male and female reproductive ducts, whereas miR449 and TAp73 complement each other to support multiciliogenesis and CP development in the brain.

Neural circuit development involves the coordinated growth and guidance of axons. During this process, axons encounter many different cues, but how these cues are integrated and translated into growth is poorly understood. In this study, we report that receptor signaling does not follow a linear path but changes dependent on developmental stage and coreceptors involved. Using developing chicken embryos of both sexes, our data show that calcium-sensing receptor (CaSR), a G-protein-coupled receptor important for regulating calcium homeostasis, regulates neurite growth in two distinct ways. First, when signaling in isolation, CaSR promotes growth through the PI3-kinase-Akt pathway. At later developmental stages, CaSR enhances tropomyosin receptor kinase B (TrkB)/BDNF-mediated neurite growth. This enhancement is facilitated through a switch in the signaling cascade downstream of CaSR (i.e., from the PI3-kinase-Akt pathway to activation of GSK3α Tyr279). TrkB and CaSR colocalize within late endosomes, cotraffic and coactivate GSK3, which serves as a shared signaling node for both receptors. Our study provides evidence that two unrelated receptors can integrate their individual signaling cascades toward a nonadditive effect and thus control neurite growth during development. SIGNIFICANCE STATEMENT This work highlights the effect of receptor coactivation and signal integration in a developmental setting. During embryonic development, neurites grow toward their targets guided by cues in the extracellular environment. These cues are sensed by receptors at the surface that trigger intracellular signaling events modulating the cytoskeleton. Emerging evidence suggests that the effects of guidance cues are diversified, therefore expanding the number of responses. Here, we show that two unrelated receptors can change the downstream signaling cascade and regulate neuronal growth through a shared signaling node. In addition to unraveling a novel signaling pathway in neurite growth, this research stresses the importance of receptor coactivation and signal integration during development of the nervous system.

Introduction: P16Ink4a is a key factor in the control of the cell cycle and governs the stem cells self-renewal and differentiation in several tissues. To investigate the role of p16 in type 2 alveolar epithelial stem cell proliferation and in lung regeneration, we used a unilateral pneumonectomy model that promotes the formation of new alveoli in the remaining lobes. Methods: Left side pneumonectomy of C57Bl/6 wild type (WT) or p16-/- mice was performed after thoracotomy, while sham mice went through thoracotomy only. Ten days after surgery morphometric analysis (mean linear intercept, alveolar surface), septal thickness, collagen deposition (Red Sirius), alveolar type 2 cells and myofibroblasts content were quantified (by immunohistochemistry for proSPC and αSMA respectively). Results: Pneumonectomised WT mice versus sham WT mice showed an increase volume of the remaining right lung, and an increase of alveolar surface but no modification of the mean linear intercept. Pneumonectomised p16-/- mice versus sham p16-/- have no modification of lung volume or alveolar surface, but increased septal thickness. In pneumonectomised group, p16-/- mice exhibited an increased number of lung cells especially alveolar type 2 cells as compared to WT mice. Collagen deposition (red Sirius) and myofibroblast content (αSMA) were not different between the groups. Conclusion: P16 deletion promotes epithelial type 2 cells differentiation during lung regeneration after pneumonectomy. P16 inhibition may be a tool to initiate and sustain alveologenesis in adults.

Planar cell polarity (PCP) and intercellular junctional complexes establish tissue structure and coordinated behaviors across epithelial sheets. In multiciliated ependymal cells, rotational and translational PCP coordinate cilia beating and direct cerebrospinal fluid circulation. Thus, PCP disruption results in ciliopathies and hydrocephalus. PCP establishment depends on the polarization of cytoskeleton and requires the asymmetric localization of core and global regulatory modules, including membrane proteins like Vangl1/2 or Frizzled. We analyzed the subcellular localization of select proteins that make up these modules in ependymal cells and the effect of Trp73 loss on their localization. We identify a novel function of the Trp73 tumor suppressor gene, the TAp73 isoform in particular, as an essential regulator of PCP through the modulation of actin and microtubule cytoskeleton dynamics, demonstrating that Trp73 is a key player in the organization of ependymal ciliated epithelia. Mechanistically, we show that p73 regulates translational PCP and actin dynamics through TAp73-dependent modulation of non-musclemyosin-II activity. In addition, TAp73 is required for the asymmetric localization of PCP-core and global signaling modules and regulates polarized microtubule dynamics, which in turn set up the rotational PCP. Therefore, TAp73 modulates, directly and/or indirectly, transcriptional programs regulating actin and microtubules dynamics and Golgi organization signaling pathways. These results shed light into the mechanism of ependymal cell planar polarization and reveal p73 as an epithelial architect during development regulating the cellular cytoskeleton.

Motile multiciliated cells (MCCs) have critical roles in respiratory health and disease and are essential for cleaning inhaled pollutants and pathogens from airways. Despite their significance for human disease, the transcriptional control that governs multiciliogenesis remains poorly understood. Here we identify TP73, a p53 homolog, as governing the program for airway multiciliogenesis. Mice with TP73 deficiency suffer from chronic respiratory tract infections due to profound defects in ciliogenesis and complete loss of mucociliary clearance. Organotypic airway cultures pinpoint TAp73 as necessary and sufficient for basal body docking, axonemal extension, and motility during the differentiation of MCC progenitors. Mechanistically, cross-species genomic analyses and complete ciliary rescue of knockout MCCs identify TAp73 as the conserved central transcriptional integrator of multiciliogenesis. TAp73 directly activates the key regulators FoxJ1, Rfx2, Rfx3, and miR34bc plus nearly 50 structural and functional ciliary genes, some of which are associated with human ciliopathies. Our results position TAp73 as a novel central regulator of MCC differentiation.

The mir-34/449 family consists of six homologous miRNAs at three genomic loci. Redundancy of miR-34/449 miRNAs and their dominant expression in multiciliated epithelia suggest a functional significance in ciliogenesis. Here we report that mice deficient for all miR-34/449 miRNAs exhibited postnatal mortality, infertility and strong respiratory dysfunction caused by defective mucociliary clearance. In both mouse and Xenopus, miR-34/449-deficient multiciliated cells (MCCs) exhibited a significant decrease in cilia length and number, due to defective basal body maturation and apical docking. The effect of miR-34/449 on ciliogenesis was mediated, at least in part, by post-transcriptional repression of Cp110, a centriolar protein suppressing cilia assembly. Consistent with this, cp110 knockdown in miR-34/449-deficient MCCs restored ciliogenesis by rescuing basal body maturation and docking. Altogether, our findings elucidate conserved cellular and molecular mechanisms through which miR-34/449 regulate motile ciliogenesis.

The microRNAs 449a, b, and c (miR-449) are potent inducers of cell death, cell cycle arrest, and/or cell differentiation. They belong to the same family as the p53-responsive microRNAs miR-34. Instead of p53, however, the cell cycle regulatory transcription factor E2F1 induces miR-449. All members of this microRNA family are capable of mediating cell cycle arrest and apoptosis and might thereby contribute to tumor suppression. Underlying mechanisms include the downregulation of histone acetyl transferases and consecutive activation of p53, but also the targeting of cyclin dependent kinases and their association partners. Thus, miR-34 and miR-449 provide an asymmetric feedback loop to balance E2F and p53 activities. More recently, it was discovered that miR-449 displays strong tissue specificity, with high levels in lung and testes. Two model systems (Xenopus embryos and cultured human cells) revealed that miR-449 is essential for the development of ciliated epithelia, and this appears to depend on miR-449-mediated modulation of the Notch signaling pathway. Here we summarize our current knowledge on cell fate determination by miR-449, and we propose future directions to explore the function of miR-449 in cell regulation and organismal development. MiR-449 helps to ensure proper cell function but also to avoid cancer, marking a close link between cell differentiation and tumor suppression.

MicroRNAs of the miR-34/449 family mediate cell cycle arrest and tumor suppression. Here we show that the expression of microRNA miR-449a, unlike its paralogue miR-34a, is highly tissue specific and largely restricted to pulmonary and testicular tissue. MiR-449a levels in the murine lung are particularly high shortly before and after birth, coinciding with terminal differentiation of lung epithelia. Strikingly, miR-449a is upregulated by more than 1000-fold when epithelial cells from human airways are lifted from a liquid environment to air, allowing them to undergo mucociliary differentiation. The induction of miR-449a occurs in parallel to its host gene CDC20B and the transcription factor FoxJ1. Exposure to tobacco smoke induces a moderate further increase in the levels of miR-449a, and also miR-34a, in differentiated airway epithelia. We propose that miR-449a can serve as an exquisitely sensitive and specific biomarker for the differentiation of bronchial epithelia. Moreover, miR-449a may actively promote mucociliary differentiation through its ability to block cell cycle progression, and it may conribute to a first line of defence against genotoxic stress by its proapoptotic functions.

EndoNet is an information resource about intercellular regulatory communication. It provides information about hormones, hormone receptors, the sources (i.e. cells, tissues and organs) where the hormones are synthesized and secreted, and where the respective receptors are expressed. The database focuses on the regulatory relations between them. An elementary communication is displayed as a causal link from a cell that secretes a particular hormone to those cells which express the corresponding hormone receptor and respond to the hormone. Whenever expression, synthesis and/or secretion of another hormone are part of this response, it renders the corresponding cell an internal node of the resulting network. This intercellular communication network coordinates the function of different organs. Therefore, the database covers the hierarchy of cellular organization of tissues and organs as it has been modeled in the Cytomer ontology, which has now been directly embedded into EndoNet. The user can query the database; the results can be used to visualize the intercellular information flow. A newly implemented hormone classification enables to browse the database and may be used as alternative entry point. EndoNet is accessible at: http://endonet.bioinf.med.uni-goettingen.de/.

E2F1 is a positive regulator of cell cycle progression and also a potent inducer of apoptosis, especially when activated by DNA damage. To identify E2F1-inducible microRNAs, I performed array hybridization and found miR-449a and miR-449b (collectively termed miR-449) to be strongly E2F1-responsive. The levels of miRNAs 449a and 449b, as well as their host gene CDC20B, are strongly upregulated by E2F1 overexpression and DNA damage. Strikingly, miR-449 shares seed sequences and target genes with the miR-34 family, which has tumour suppressive properties. MiR-449 is expressed at high levels and very specifically in testes, lung, and trachea, but not in tumour cells. However, the expression of miR-449 can be reactivated in tumour cells lines by HDAC inhibition, suggesting epigenetic silencing in cancer. Furthermore, miR-449 expression is strongly induced during the mucociliary differentiation of pulmonary epithelia. Exposure to tobacco smoke further increases the levels of miR-449 in airways, consistent with its DNA damage responsiveness. Therefore, miR-449 can serve as an exquisitely sensitive and specific biomarker for the differentiation of mucociliary epithelium. Moreover, it may actively promote differentiation through its ability to block cell cycle progression, and provide a first line of defence against genotoxic stress by its proapoptotic functions.In agreement with a putative tumour-suppressive role, miR-449 as well as miR-34 reduce proliferation, upregulate p53 activity and strongly promote apoptosis through both p53-dependent and -independent mechanisms. Both miRNAs attenuate E2F1 function by reducing the levels of CDK6, CDK2, E2F1 and E2F3, implying a negative feedback mechanism for the E2F pathway. Moreover, miR-449 and miR-34 decrease the expression of the deacetylases SIRT1 and HDAC1, thereby resulting in p53 activation, which in turn leads to p21 induction and stronger cell cycle arrest in p53 wild type cells. Moreover, since histone deacetylases can regulate complete gene expression programmes, the down-regulation of HDAC1 and SIRT1 might dramatically change the expression patterns, and therefore influence cell fate. In fact, HDAC inhibition has been shown to be very effective in cancer therapy. In addition, miR-449 and miR-34 downregulate two important cell cycle checkpoint proteins, Chk1 and BRCA1, and this can lead to cell death through mitotic catastrophe by interfering with normal mitotic checkpoint regulation. Thus, miR-449 can induce apoptosis in tumour cells in a p53-independent manner.While E2F1-induced miR-449 as well as p53-induced miR-34 promote p53 activity and apoptosis, they negatively regulate E2F1. Hence, the influence of E2F1 and p53 on each other and on cell fate decisions is sustained by the induction of two miRNA species from the same family. Both miR-449 and miR-34 could provide a twofold safety mechanism to avoid excessive E2F1-induced proliferation by leading to cell cycle arrest or apoptosis.