Progressive tissue fibrosis, including idiopathic pulmonary fibrosis (IPF), is characterized by excessive recruitment of fibroblasts to sites of tissue injury and unremitting extracellular matrix ...deposition associated with severe morbidity and mortality. However, the molecular mechanisms that control progressive IPF have yet to be fully determined. Previous studies suggested that invasive fibroblasts drive disease progression in IPF. Here, we report profiling of invasive and noninvasive fibroblasts from IPF patients and healthy donors. Pathway analysis revealed that the activated signatures of the invasive fibroblasts, the top of which was ERBB2 (HER2), showed great similarities to those of metastatic lung adenocarcinoma cancer cells. Activation of HER2 in normal lung fibroblasts led to a more invasive genetic program and worsened fibroblast invasion and lung fibrosis, while antagonizing HER2 signaling blunted fibroblast invasion and ameliorated lung fibrosis. These findings suggest that HER2 signaling may be a key driver of fibroblast invasion and serve as an attractive target for therapeutic intervention in IPF.
Loss of epithelial integrity, bronchiolarization, and fibroblast activation are key characteristics of idiopathic pulmonary fibrosis (IPF). Prolonged accumulation of basal-like cells in IPF may ...impact the fibrotic niche to promote fibrogenesis. To investigate their role in IPF, basal cells were isolated from IPF explant and healthy donor lung tissues. Single-cell RNA sequencing was used to assess differentially expressed genes in basal cells. Basal cell and niche interaction was demonstrated with the sLP-mCherry niche labeling system. Luminex assays were used to assess cytokines secreted by basal cells. The role of basal cells in fibroblast activation was studied. Three-dimensional organoid culture assays were used to interrogate basal cell effects on AEC2 (type 2 alveolar epithelial cell) renewal capacity. Perturbation was used to investigate WNT7A function
and in a repetitive bleomycin model
. We found that WNT7A is highly and specifically expressed in basal-like cells. Proteins secreted by basal cells can be captured by neighboring fibroblasts and AEC2s. Basal cells or basal cell-conditioned media activate fibroblasts through WNT7A. Basal cell-derived WNT7A inhibits AEC2 progenitor cell renewal in three-dimensional organoid cultures. Neutralizing antibodies against WNT7A or a small molecule inhibitor of Frizzled signaling abolished basal cell-induced fibroblast activation and attenuated lung fibrosis in mice. In summary, basal cells and basal cell-derived WNT7A are key components of the fibrotic niche, providing a unique non-stem cell function of basal cells in IPF progression and a novel targeting strategy for IPF.
Mechanisms that regulate progenitor cell quiescence and differentiation in slowly replacing tissues are not fully understood. Here, we demonstrate that the tumor suppressor p53 regulates both ...proliferation and differentiation of progenitors in the airway epithelium. p53 loss decreased ciliated cell differentiation and increased the self-renewal and proliferative capacity of club progenitors, increasing epithelial cell density. p53-deficient progenitors generated a pseudostratified epithelium containing basal-like cells in vitro and putative bronchioalveolar stem cells in vivo. Conversely, an additional copy of p53 increased quiescence and ciliated cell differentiation, highlighting the importance of tight regulation of p53 levels. Using single-cell RNA sequencing, we found that loss of p53 altered the molecular phenotype of progenitors and differentially modulated cell-cycle regulatory genes. Together, these findings reveal that p53 is an essential regulator of progenitor cell behavior, which shapes our understanding of stem cell quiescence during homeostasis and in cancer development.
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•p53 regulates proliferation and differentiation of airway epithelial progenitors•Loss of p53 increases progenitor self-renewal, multipotency, and proliferation•An extra copy of p53 promotes terminal differentiation and decreased proliferation•Single-cell RNA-seq reveals that p53 alters molecular phenotype of progenitors
Through genetic manipulation of p53 copy number in vivo and in vitro, McConnell et al. show that p53 is a key regulator of airway epithelial cell density and composition. This is due to the ability of p53 to control proliferation and differentiation of progenitor cells.
Context: Crosstalk through receptor ligand interactions at the maternal-fetal interface is impacted by fetal sex. This affects placentation in the first trimester and differences in outcomes. ...Sexually dimorphic signaling at early stages of placentation are not defined. Objective: Investigate the impact of fetal sex on maternal-fetal crosstalk. Design: Receptors/ligands at the maternal-fetal surface were identified from sexually dimorphic genes between fetal sexes in the first trimester placenta and defined in each cell type using single-cell RNA-Sequencing (scRNA-Seq). Setting: Academic institution. Samples: Late first trimester (~10-13 weeks) placenta (fetal) and decidua (maternal) from uncomplicated ongoing pregnancies. Main outcome measures: Transcriptomic profiling at tissue and single-cell level; immunohistochemistry of select proteins. Results: We identified 91 sexually dimorphic receptor-ligand pairs across the maternalfetal interface. We examined fetal sex differences in 5 major cell types (trophoblasts, stromal cells, Hofbauer cells, antigen-presenting cells, and endothelial cells). Ligands from the CC family chemokine ligand (CCL) family were most highly representative in females, with their receptors present on the maternal surface. Sexually dimorphic trophoblast transcripts, Mucin-15 (MUC15) and notum, palmitoleoyl-protein carboxylesterase (NOTUM) were also most highly expressed in syncytiotrophoblasts and extravillous trophoblasts respectively. Gene Ontology (GO) analysis using sexually dimorphic genes in individual cell types identified cytokine mediated signaling pathways to be most representative in female trophoblasts. Upstream analysis demonstrated TGFB1 and estradiol to affect all cell types, but dihydrotestosterone, produced by the male fetus, was an upstream regulator most significant for the trophoblast population. Conclusions: Maternal-fetal crosstalk exhibits sexual dimorphism during placentation early in gestation. Key Words: first trimester placenta, sex differences, human pregnancy, single-cell RNA sequencing, placenta cell types, receptor-ligand
The functional properties of mucosal surfaces are dependent on establishing the correct proportions of specialized epithelial cell types. Multiciliated cells (also known as ciliated cells) are ...evolutionarily conserved and functionally indispensable epithelial cells, as suggested by the link between ciliated cell dysfunction and chronic human disease. Ciliated cell differentiation is an ordered process that involves initial cell fate determination and multiciliogenesis. STK11, a serine/threonine kinase, has been reported to be downregulated in human diseases associated with ciliopathies and functions as a tumor suppressor. Here, we show that STK11 is a physiological factor for the normal program of ciliated cell differentiation by phosphorylating MARK3, which directly suppresses ERK1/2 mediated pRB inactivation. Loss of
in airway progenitors impairs the differentiation of ciliated cells in both embryonic and adult airways. Our study establishes that STK11/MARK3/ERK1/2 signaling cascade is a key regulator to integrate ciliated cell fate commitment and the subsequent process of multiciliogenesis.
Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease. A maladaptive epithelium due to chronic injury is a prominent feature and contributor to pathogenic cellular communication in ...IPF. Recent data highlight the concept of a "reprogrammed" lung epithelium as critical in the development of lung fibrosis. Extracellular vesicles (EVs) are potent mediator of cellular crosstalk, and recent evidence supports their role in lung pathologies such as IPF. Here, we demonstrate that syndecan-1 is overexpressed by the epithelium in the lungs of IPF patients and in murine models after bleomycin injury. Moreover, we find that syndecan-1 is a pro-fibrotic signal that alters alveolar type II (ATII) cell phenotypes by augmenting TGFβ and Wnt signaling among other pro-fibrotic pathways. Importantly, we demonstrate that syndecan-1 controls the packaging of several anti-fibrotic microRNAs into EVs that have broad effects over several fibrogenic signaling networks as a mechanism of regulating epithelial plasticity and pulmonary fibrosis. Collectively, our work reveals new insight into how EVs orchestrate cellular signals that promote lung fibrosis and demonstrate the importance of syndecan-1 in coordinating these programs.
The idea of a spindle matrix has long been proposed in order to account for poorly understood features of mitosis. However, its molecular nature and structural composition have remained elusive. ...Here, we propose that the spindle matrix may be constituted by mainly nuclear-derived proteins that reorganize during the cell cycle to form an elastic gel-like matrix. We discuss this hypothesis in the context of recent observations from phylogenetically diverse organisms that nuclear envelope and intranuclear proteins form a highly dynamic and malleable structure that contributes to mitotic spindle function. We suggest that the viscoelastic properties of such a matrix may constrain spindle length while at the same time facilitating microtubule growth and dynamics as well as chromosome movement. A corollary to this hypothesis is that a key determinant of spindle size may be the amount of nuclear proteins available to form the spindle matrix. Such a matrix could also serve as a spatial regulator of spindle assembly checkpoint proteins during open and semi-open mitosis.
Recent studies have identified impaired type 2 alveolar epithelial cell (ATII) renewal in idiopathic pulmonary fibrosis (IPF) human organoids and severe fibrosis when ATII is defective in mice. ATIIs ...function as progenitor cells and require supportive signals from the surrounding mesenchymal cells. The mechanisms by which mesenchymal cells promote ATII progenitor functions in lung fibrosis are incompletely understood. We identified growth hormone receptor (GHR) is mainly expressed in mesenchymal cells, and its expression is substantially decreased in IPF lungs. Higher levels of
expression correlated with better lung function in patients with IPF. Profibrotic mesenchymal cells retarded ATII growth and were associated with suppressed vesicular
expression. Vesicles enriched with
promote ATII proliferation and diminished pulmonary fibrosis in mesenchymal
-deficient mice. Our findings demonstrate a previously unidentified mesenchymal paracrine signaling coordinated by
that is capable of supporting ATII progenitor cell renewal and limiting the severity of lung fibrosis.
The JIL-1 kinase localizes to Drosophila polytene chromosome interbands and phosphorylates histone H3 at interphase, counteracting histone H3 lysine 9 dimethylation and gene silencing. JIL-1 can be ...divided into four main domains, including an NH2-terminal domain, two separate kinase domains, and a COOH-terminal domain. In this study, we characterize the domain requirements of the JIL-1 kinase for histone H3 serine 10 (H3S10) phosphorylation and chromatin remodeling in vivo. We show that a JIL-1 construct without the NH2-terminal domain is without H3S10 phosphorylation activity despite the fact that it localizes properly to polytene interband regions and that it contains both kinase domains. JIL-1 is a double kinase, and we demonstrate that both kinase domains of JIL-1 are required to be catalytically active for H3S10 phosphorylation to occur. Furthermore, we provide evidence that JIL-1 is phosphorylated at serine 424 and that this phosphorylation is necessary for JIL-1 H3S10 phosphorylation activity. Thus, these data are compatible with a model where the NH2-terminal domain of JIL-1 is required for chromatin complex interactions that position the kinase domain(s) for catalytic activity in the context of the state of higher order nucleosome packaging and chromatin structure and where catalytic H3S10 phosphorylation activity mediated by the first kinase domain is dependent on autophosphorylation of serine 424 by the second kinase domain. Furthermore, using a lacO repeat tethering system to target mutated JIL-1 constructs with or without catalytic activity, we show that the epigenetic H3S10 phosphorylation mark itself functions as a causative regulator of chromatin structure independently of any structural contributions from the JIL-1 protein.
Background: The JIL-1 histone H3 serine 10 (H3S10) tandem kinase functions to maintain active gene expression.
Results: The NH2-terminal domain and both kinase domains are required for H3S10 kinase activity.
Conclusion: The NH2-terminal domain is required for chromatin complex interactions, and H3S10 phosphorylation functions as a causative regulator of chromatin structure.
Significance: Determining how the domain structure of JIL-1 contributes to its function is crucial for understanding regulation of gene expression.
The chromodomain protein, Chromator, has been shown to have multiple functions that include regulation of chromatin structure as well as coordination of muscle remodeling during metamorphosis ...depending on the developmental context. In this study we show that mitotic neuroblasts from brain squash preparations from larvae heteroallelic for the two
Chromator loss-of-function alleles
Chro
71
and
Chro
612
have severe microtubule spindle and chromosome segregation defects that were associated with a reduction in brain size. The microtubule spindles formed were incomplete, unfocused, and/or without clear spindle poles and at anaphase chromosomes were lagging and scattered. Time-lapse analysis of mitosis in S2 cells depleted of Chromator by RNAi treatment suggested that the lagging and scattered chromosome phenotypes were caused by incomplete alignment of chromosomes at the metaphase plate, possibly due to a defective spindle-assembly checkpoint, as well as of frayed and unstable microtubule spindles during anaphase. Expression of full-length Chromator transgenes under endogenous promoter control restored both microtubule spindle morphology as well as brain size strongly indicating that the observed mutant defects were directly attributable to lack of Chromator function.
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