Identification of Meibomian gland stem cell populations and mechanisms of aging.

Meibomian glands secrete lipid-rich meibum, which prevents tear evaporation. Aging-related Meibomian gland shrinkage may result in part from stem cell exhaustion and is associated with evaporative dry eye disease, a common condition lacking effective treatment. The identities and niche of Meibomian gland stem cells and the signals controlling their activity are poorly defined. Using snRNA-seq, in vivo lineage tracing, ex vivo live imaging, and genetic studies in mice, we identified markers for stem cell populations that maintain distinct regions of the gland and uncovered Hh signaling as a key regulator of stem cell proliferation. Consistent with this, human Meibomian gland carcinoma exhibited increased Hh signaling. Aged glands displayed decreased Hh and EGF signaling, deficient innervation, and loss of collagen I in niche fibroblasts, indicating that alterations in both glandular epithelial cells and their surrounding microenvironment contribute to age-related degeneration. These findings suggest new approaches to treat aging-associated Meibomian gland loss.

HDAC1/2 and HDAC3 play distinct roles in controlling adult Meibomian gland homeostasis.

PURPOSE: To investigate the roles of HDAC1/2 and HDAC3 in adult Meibomian gland (MG) homeostasis. METHODS: HDAC1/2 or HDAC3 were inducibly deleted in MG epithelial cells of adult mice. The morphology of MG was examined. Proliferation, apoptosis, and expression of MG acinus and duct marker genes, meibocyte differentiation genes, and HDAC target genes, were analyzed via immunofluorescence, TUNEL assay, and RNA in situ hybridization. RESULTS: Co-deletion of HDAC1/2 in MG epithelium caused gradual loss of acini and formation of cyst-like structures in the central duct. These phenotypes required homozygous deletion of both HDAC1 and HDAC2, indicating that they function redundantly in the adult MG. Short-term deletion of HDAC1/2 in MG epithelium had little effect on meibocyte maturation but caused decreased proliferation of acinar basal cells, excessive DNA damage, ectopic apoptosis, and increased p53 acetylation and p16 expression in the MG. By contrast, HDAC3 deletion in MG epithelium caused dilation of central duct, atrophy of acini, defective meibocyte maturation, increased acinar basal cell proliferation, and ectopic apoptosis and DNA damage. Levels of p53 acetylation and p21 expression were elevated in HDAC3-deficient MGs, while the expression of the differentiation regulator PPARγ and the differentiation markers PLIN2 and FASN was downregulated. CONCLUSIONS: HDAC1 and HDAC2 function redundantly in adult Meibomian gland epithelial progenitor cells and are essential for their proliferation and survival, but not for acinar differentiation, while HDAC3 is required to limit acinar progenitor cell proliferation and permit differentiation. HDAC1/2 and HDAC3 have partially overlapping roles in maintaining survival of MG cells.

FZD2 regulates limb development by mediating ß-catenin-dependent and -independent Wnt signaling pathways.

Human Robinow syndrome (RS) and dominant omodysplasia type 2 (OMOD2), characterized by skeletal limb and craniofacial defects, are associated with heterozygous mutations in the Wnt receptor FZD2. However, as FZD2 can activate both canonical and non-canonical Wnt pathways, its precise functions and mechanisms of action in limb development are unclear. To address these questions, we generated mice harboring a single-nucleotide insertion in Fzd2 (Fzd2em1Smill), causing a frameshift mutation in the final Dishevelled-interacting domain. Fzd2em1Smill mutant mice had shortened limbs, resembling those of RS and OMOD2 patients, indicating that FZD2 mutations are causative. Fzd2em1Smill mutant embryos displayed decreased canonical Wnt signaling in developing limb mesenchyme and disruption of digit chondrocyte elongation and orientation, which is controlled by the ß-catenin-independent WNT5A/planar cell polarity (PCP) pathway. In line with these observations, we found that disruption of FZD function in limb mesenchyme caused formation of shortened bone elements and defects in Wnt/ß-catenin and WNT5A/PCP signaling. These findings indicate that FZD2 controls limb development by mediating both canonical and non-canonical Wnt pathways and reveal causality of pathogenic FZD2 mutations in RS and OMOD2 patients.

Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection.

Angiotensin-converting enzyme 2 (ACE2) is the cell-surface receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). While its central role in Coronavirus Disease 2019 (COVID-19) pathogenesis is indisputable, there remains significant debate regarding the role of this transmembrane carboxypeptidase in the disease course. These include the role of soluble versus membrane-bound ACE2, as well as ACE2-independent mechanisms that may contribute to viral spread. Testing these roles requires in vivo models. Here, we report humanized ACE2-floxed mice in which hACE2 is expressed from the mouse Ace2 locus in a manner that confers lethal disease and permits cell-specific, Cre-mediated loss of function, and LSL-hACE2 mice in which hACE2 is expressed from the Rosa26 locus enabling cell-specific, Cre-mediated gain of function. Following exposure to SARS-CoV-2, hACE2-floxed mice experienced lethal cachexia, pulmonary infiltrates, intravascular thrombosis and hypoxemia-hallmarks of severe COVID-19. Cre-mediated loss and gain of hACE2 demonstrate that neuronal infection confers lethal cachexia, hypoxemia, and respiratory failure in the absence of lung epithelial infection. In this series of genetic experiments, we demonstrate that ACE2 is absolutely and cell-autonomously required for SARS-CoV-2 infection in the olfactory epithelium, brain, and lung across diverse cell types. Therapies inhibiting or blocking ACE2 at these different sites are likely to be an effective strategy towards preventing severe COVID-19.

Author Correction: MiR-31 promotes mammary stem cell expansion and breast tumorigenesis by suppressing Wnt signaling antagonists.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

HDAC3 ensures stepwise epidermal stratification via NCoR/SMRT-reliant mechanisms independent of its histone deacetylase activity.

Chromatin modifiers play critical roles in epidermal development, but the functions of histone deacetylases in this context are poorly understood. The class I HDAC, HDAC3, is of particular interest because it plays divergent roles in different tissues by partnering with tissue-specific transcription factors. We found that HDAC3 is expressed broadly in embryonic epidermis and is required for its orderly stepwise stratification. HDAC3 protein stability in vivo relies on NCoR and SMRT, which function redundantly in epidermal development. However, point mutations in the NCoR and SMRT deacetylase-activating domains, which are required for HDAC3's enzymatic function, permit normal stratification, indicating that HDAC3's roles in this context are largely independent of its histone deacetylase activity. HDAC3-bound sites are significantly enriched for predicted binding motifs for critical epidermal transcription factors including AP1, GRHL, and KLF family members. Our results suggest that among these, HDAC3 operates in conjunction with KLF4 to repress inappropriate expression of Tgm1, Krt16, and Aqp3 In parallel, HDAC3 suppresses expression of inflammatory cytokines through a Rela-dependent mechanism. These data identify HDAC3 as a hub coordinating multiple aspects of epidermal barrier acquisition.

Cytoplasmic chromatin triggers inflammation in senescence and cancer.

Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

MiR-31 promotes mammary stem cell expansion and breast tumorigenesis by suppressing Wnt signaling antagonists.

MicroRNA-mediated post-transcriptional regulation plays key roles in stem cell self-renewal and tumorigenesis. However, the in vivo functions of specific microRNAs in controlling mammary stem cell (MaSC) activity and breast cancer formation remain poorly understood. Here we show that miR-31 is highly expressed in MaSC-enriched mammary basal cell population and in mammary tumors, and is regulated by NF-κB signaling. We demonstrate that miR-31 promotes mammary epithelial proliferation and MaSC expansion at the expense of differentiation in vivo. Loss of miR-31 compromises mammary tumor growth, reduces the number of cancer stem cells, as well as decreases tumor-initiating ability and metastasis to the lung, supporting its pro-oncogenic function. MiR-31 modulates multiple signaling pathways, including Prlr/Stat5, TGFß and Wnt/ß-catenin. Particularly, it activates Wnt/ß-catenin signaling by directly targeting Wnt antagonists, including Dkk1. Importantly, Dkk1 overexpression partially rescues miR31-induced mammary defects. Together, these findings identify miR-31 as the key regulator of MaSC activity and breast tumorigenesis.

WNT10A mutation causes ectodermal dysplasia by impairing progenitor cell proliferation and KLF4-mediated differentiation.

Human WNT10A mutations are associated with developmental tooth abnormalities and adolescent onset of a broad range of ectodermal defects. Here we show that ß-catenin pathway activity and adult epithelial progenitor proliferation are reduced in the absence of WNT10A, and identify Wnt-active self-renewing stem cells in affected tissues including hair follicles, sebaceous glands, taste buds, nails and sweat ducts. Human and mouse WNT10A mutant palmoplantar and tongue epithelia also display specific differentiation defects that are mimicked by loss of the transcription factor KLF4. We find that ß-catenin interacts directly with region-specific LEF/TCF factors, and with KLF4 in differentiating, but not proliferating, cells to promote expression of specialized keratins required for normal tissue structure and integrity. Our data identify WNT10A as a critical ligand controlling adult epithelial proliferation and region-specific differentiation, and suggest downstream ß-catenin pathway activation as a potential approach to ameliorate regenerative defects in WNT10A patients.

Beta-catenin is essential for ameloblast movement during enamel development.

Beta-catenin is a multifunctional protein that plays key roles in cadherin-based cell adherens junctions and in the Wnt signaling pathway. The canonical Wnt/ß-catenin pathway can regulate transcription factors that control cell movement/invasion. We investigated whether ß-catenin regulates ameloblast movement through canonical Wnt signaling. The morphological and physical properties of enamel were assessed in enamel from control and ß-catenin conditional knockout (cKO) mice. Ameloblast-lineage cells (ALC) were used to investigate the potential roles of ß-catenin in cell migration and in E-cadherin expression. Compared with controls, incisors from ß-catenin cKO mice were short, blunt, and where enamel was present, it was soft and malformed. Scanning electron microscopy revealed a dysplastic rod pattern within the enamel of incisors from ß-catenin cKO mice, and Vickers microhardness measurements confirmed that mice with ß-catenin ablated from their enamel organ had enamel that was significantly softer than normal. Amelogenesis was disrupted in the absence of ß-catenin and the ameloblasts did not differentiate properly. We further demonstrated that migration of ALCs was inhibited in vitro and that E-cadherin expression was significantly up-regulated when ALCs were treated with the ß-catenin inhibitor, ICG-001. Beta-catenin ablation causes enamel malformation in mice and this phenotype may occur, in part, by a lack of ameloblast differentiation and/or movement necessary to form the decussating enamel rod structure.

ß-Catenin Signaling Biases Multipotent Lingual Epithelial Progenitors to Differentiate and Acquire Specific Taste Cell Fates.

Continuous taste bud cell renewal is essential to maintain taste function in adults; however, the molecular mechanisms that regulate taste cell turnover are unknown. Using inducible Cre-lox technology, we show that activation of ß-catenin signaling in multipotent lingual epithelial progenitors outside of taste buds diverts daughter cells from a general epithelial to a taste bud fate. Moreover, while taste buds comprise 3 morphological types, ß-catenin activation drives overproduction of primarily glial-like Type I taste cells in both anterior fungiform (FF) and posterior circumvallate (CV) taste buds, with a small increase in Type II receptor cells for sweet, bitter and umami, but does not alter Type III sour detector cells. Beta-catenin activation in post-mitotic taste bud precursors likewise regulates cell differentiation; forced activation of ß-catenin in these Shh+ cells promotes Type I cell fate in both FF and CV taste buds, but likely does so non-cell autonomously. Our data are consistent with a model where ß-catenin signaling levels within lingual epithelial progenitors dictate cell fate prior to or during entry of new cells into taste buds; high signaling induces Type I cells, intermediate levels drive Type II cell differentiation, while low levels may drive differentiation of Type III cells.

Polypyrimidine tract-binding protein 2 binds to selective, intronic messenger RNA and microRNA targets in the mouse testis.

Here we use an in vivo cross-linking and immunoprecipitation procedure to detect RNA targets of the multifunctional RNA-binding protein polypyrimidine tract-binding protein (PTBP) 2 in mouse testis. Eleven known mRNAs, including Ptbp2 mRNA, 28 RNAs matching intron sequences, and 12 small RNAs and repeat sequences are identified. The specificity of interaction between PTBP2 and its target RNAs was confirmed using RNA interference with mouse N2A cells. Reduction of PTBP2 levels led to decreases in 7 of 10 of the mRNAs, to the repression of alternative splicing of introns, and to reductions in specific miRNAs.

A cytoplasmic variant of the KH-type splicing regulatory protein serves as a decay-promoting factor for phosphoglycerate kinase 2 mRNA in murine male germ cells.

Phosphoglycerate kinase 2 (PGK2) is a germ cell-specific protein whose mRNA is translationally regulated in the mammalian testis. Using RNA affinity chromatography with the 3'-untranslated region (UTR) of Pgk2 mRNA and adult testis extracts, several associated proteins including a novel isoform of the AU-rich element RNA-binding protein and KH-type splicing regulatory protein (KSRP) were identified. KSRP, a protein of approximately 75 kDa, is widely expressed in somatic and germ cells where it is primarily nuclear. In addition to the approximately 75-kDa KSRP, a approximately 52-kD KSRP, t-KSRP, is present in the cytoplasm of a subpopulation of germ cells. t-KSRP binds directly to a 93-nt sequence (designated the F1 region) of the 3'-UTR of the Pgk2 mRNA and destabilizes Pgk2 mRNA constructs in testis extracts and in transfected cells. We conclude that this testicular variant of the multifunctional nucleic acid-binding protein, KSRP, serves as a decay-promoting factor for Pgk2 mRNA in male germ cells.

MSY2 and polypyrimidine tract binding protein 2 stabilize mRNAs in the mammalian testis.

MSY2 is a highly conserved and abundant DNA/RNA-binding protein that functions as a global stabilizer/translational suppressor of mRNAs in male germ cells. The polypyrimidine tract binding protein, PTBP2, is an RNA-binding protein that splices nuclear transcripts and stabilizes specific mRNAs in the cytoplasm. The mechanisms whereby MSY2 selects and stabilizes a large group of male germ cell mRNAs and PTBP2 stabilizes specific mRNAs such as the phosphoglycerate kinase 2 mRNA in the testis and in transfected cells will be discussed.

Polypyrimidine tract binding protein 2 stabilizes phosphoglycerate kinase 2 mRNA in murine male germ cells by binding to its 3'UTR.

The mRNA that encodes the testis-specific protein phosphoglycerate kinase (PGK2) is a long-lived mRNA that is transcribed in meiotic and postmeiotic male germ cells. Pgk2 mRNA is present in germ cells for up to 2 wk before its protein product is detected. Using affinity chromatography with the 3'-UTR of the Pgk2 mRNA, several proteins, including the RNA-binding protein, polypyrimidine tract binding protein 2 (PTBP2), were identified in mouse testis extracts. Coimmunoprecipitation experiments confirmed that PTBP2 binds to Pgk2 mRNA in the testis and RNA gel shifts demonstrated that PTBP2, but not PTBP1, binds to a specific region of the Pgk2 3'-UTR. Recombinant PTBP2 increased the stability of reporter constructs that contained the 3'-UTR Pgk2 sequence element in both testis extracts and transfected HeLa cells. We propose that PTBP2 is a trans-acting factor that helps to stabilize Pgk2 mRNA in male mouse germ cells.

Two forms of human cytoplasmic arginyl-tRNA synthetase produced from two translation initiations by a single mRNA.

Human cytoplasmic arginyl-tRNA synthetase (ArgRS) is a component of a macromolecular complex consisting of at least nine tRNA synthetases and three auxiliary proteins. In mammalian cells, ArgRS is present as a free protein as well as a component of the complex. Via an alignment of ArgRSs from different vertebrates, the genes encoding full-length human cytoplasmic ArgRS and an N-terminal 72-amino acid deletion mutant (hcArgRS and DeltaNhcArgRS, respectively) were subcloned and expressed in Escherichia coli. The two ArgRS products were expressed as a soluble protein in E. coli. The level of production of DeltaNhcArgRS in E. coli and its specific activity were higher than those for hcArgRS. By Western blot analysis, using an antibody against the purified DeltaNhcArgRS, the two forms of ArgRS were detected in three human cell types. The 5'-end cDNA sequence, as confirmed by 5'RACE (5'-rapid amplification of cDNA ends), contained three start codons. Through mutation of the three codons, the two human cytoplasmic ArgRSs were found to be produced in different amounts, indicating that they resulted from two different translation initiation events. Here we show evidence that two forms of human cytoplasmic ArgRS were produced from two translational initiations by a single mRNA.

Leucyl-tRNA synthetase from the hyperthermophilic bacterium Aquifex aeolicus recognizes minihelices.

Aminoacylation of the minihelix mimicking the amino acid acceptor arm of tRNA has been demonstrated in more than 10 aminoacyl-tRNA synthetase systems. Although Escherichia coli or Homo sapiens cytoplasmic leucyl-tRNA synthetase (LeuRS) is unable to charge the cognate minihelix or microhelix, we show here that minihelix(Leu) is efficiently charged by Aquifex aeolicus synthetase, the only known heterodimeric LeuRS (alpha beta-LeuRS). Aminoacylation of minihelices is strongly dependent on the presence of the A73 identity nucleotide and greatly stimulated by destabilization of the first base pair as reported for the E. coli isoleucyl-tRNA synthetase and methionyl-tRNA synthetase systems. In the E. coli LeuRS system, the anticodon of tRNA(Leu) is not important for recognition by the synthetase. However, the addition of RNA helices that mimic the anticodon domain stimulates minihelix(Leu) charging by alpha beta-LeuRS, indicating possible domain-domain communication within alpha beta-LeuRS. The leucine-specific domain of alpha beta-LeuRS is responsible for minihelix recognition. To ensure accurate translation of the genetic code, LeuRS functions to hydrolyze misactivated amino acids (pretransfer editing) and misaminoacylated tRNA (posttransfer editing). In contrast to tRNA(Leu), minihelix(Leu) is unable to induce posttransfer editing even upon the addition of the anticodon domain of tRNA. Therefore, the context of tRNA is crucial for the editing of mischarged products. However, the minihelix(Leu) cannot be misaminoacylated, perhaps because of the tRNA-independent pretransfer editing activity of alpha beta-LeuRS.

Groups on the side chain of T252 in Escherichia coli leucyl-tRNA synthetase are important for discrimination of amino acids and cell viability.

Leucyl-tRNA synthetase (LeuRS) catalyzes the leucylation of tRNA(Leu). To maintain the fidelity of protein biosynthesis, LeuRS also catalyzes the editing reaction. In the present work, highly conserved T252 in the T-rich region within CP1 domain of Escherichia coli LeuRS was mutated to G, D, or E. Steady-state kinetic of aminoacylation, and combined editing assays indicated that not only the size of the amino acid but also the absence of hydrogen bonds between T252 and adjacent molecules may affect the editing. It is further confirmed by in vivo experiments using the temperature-sensitive strain KL231 (DeltaleuS), which revealed the arrested growth of bacterial cells bearing mutants with highly impaired editing activity in the presence of leucine analog.

Leucyl-tRNA synthetase consisting of two subunits from hyperthermophilic bacteria Aquifex aeolicus.

In a hyperthermophilic bacterium, Aquifex aeolicus, leucyl-tRNA synthetase (LeuRS) consists of two non-identical polypeptide subunits (alpha and beta), different from the canonical LeuRS, which has a single polypeptide chain. By PCR, using genome DNA of A. aeolicus as a template, genes encoding the alpha and beta subunits were amplified and cloned in Escherichia coli. The alpha subunit could not be expressed stably in vivo, whereas the beta subunit was overproduced and purified by a simple procedure. The beta subunit was inactive in catalysis but was able to bind tRNA(Leu). Interestingly, the heterodimer alphabeta-LeuRS could be overproduced in E. coli cells containing both genes and was purified to 95% homogeneity as a hybrid dimer. The kinetics of A. aeolicus LeuRS in pre-steady and steady states and cross-recognition of LeuRS and tRNA(Leu) from A. aeolicus and E. coli were studied. Magnesium concentration, pH value, and temperature aminoacylation optima were determined to be 12 mm, 7.8, and 70 degrees C, respectively. Under optimal conditions, A. aeolicus alphabeta-LeuRS is stable up to 65 degrees C.