Very little is known about the in vivo regulation of mammalian fatty acid chain elongation enzymes as well as the role of specific fatty acid chain length in cellular responses and developmental processes. Here, we report that the Elovl3 gene product, which belongs to a highly conserved family of microsomal enzymes involved in the formation of very long chain fatty acids, revealed a distinct expression in the skin that was restricted to the sebaceous glands and the epithelial cells of the hair follicles. By disruption of the Elovl3 gene by homologous recombination in mouse, we show that ELOVL3 participates in the formation of specific neutral lipids that are necessary for the function of the skin. The Elovl3-ablated mice displayed a sparse hair coat, the pilosebaceous system was hyperplastic, and the hair lipid content was disturbed with exceptionally high levels of eicosenoic acid (20:1). This was most prominent within the triglyceride fraction where fatty acids longer than 20 carbon atoms were almost undetectable. A functional consequence of this is that Elovl3-ablated mice exhibited a severe defect in water repulsion and increased trans-epidermal water loss.Fatty acids consisting of up to 16 carbons are synthesized by the well studied fatty acid synthase complex (1). However, a significant amount of the fatty acids produced by fatty acid synthase are further elongated into very long chain fatty acids (VLCFA). 1 VLCFA have been recognized as structural components in a variety of fat molecules such as glycerolipids and sphingolipids. They are found in virtually all cells and are major constituents of the brain, skin, and testis (2-4). Depending on their chain length and degree of unsaturation, they contribute to membrane fluidity and other chemical properties of the cell.Formation of VLCFA is performed in the endoplasmic reticulum, in the early Golgi, and in mitochondria by membranebound enzymes, the former being more prominent (5, 6).Recently, five mammalian genes, Elovl1-5, 2 whose protein products belong to a highly conserved family of microsomal enzymes involved in the formation of VLCFA, have been identified (7-10). All five genes show a diverse tissue-specific expression pattern indicating a unique role for different VLCFA in different cell types.Although the general functions of the Elovl genes are partially understood (8 -14), very little is known about the role of specific fatty acid chain length in cellular responses and developmental processes. The ELOVL3 protein has been suggested to be involved in the formation of saturated and monounsaturated fatty acyl chains containing up to 24 carbon atoms (9). Elovl3 gene expression has only been detected in brown adipose tissue, liver, and skin (7, 9). To assess the in vivo role of ELOVL3, we disrupted the Elovl3 gene by homologous recombination in mouse. Here we describe the characterization of Elovl3 expression in skin and present evidence that ELOVL3 participates in the formation of certain VLCFA and triglycerides in certain cells of the hair follicles and...
The hydroxymethyl group of serine is a primary source of tetrahydrofolate (THF)-activated one-carbon units that are required for the synthesis of purines and thymidylate and for S-adenosylmethionine (AdoMet)-dependent methylation reactions. Serine hydroxylmethyltransferase (SHMT) catalyzes the reversible and THF-dependent conversion of serine to glycine and 5,10-methylene-THF. SHMT is present in eukaryotic cells as mitochondrial SHMT and cytoplasmic (cSHMT) isozymes that are encoded by distinct genes. In this study, the essentiality of cSHMT-derived THF-activated one-carbons was investigated by gene disruption in the mouse germ line. Mice lacking cSHMT are viable and fertile, demonstrating that cSHMT is not an essential source of THF-activated one-carbon units. cSHMTdeficient mice exhibit altered hepatic AdoMet levels and uracil content in DNA, validating previous in vitro studies that indicated this enzyme regulates the partitioning of methylenetetrahydrofolate between the thymidylate and homocysteine remethylation pathways. This study suggests that mitochondrial SHMT-derived one-carbon units are essential for folate-mediated one-carbon metabolism in the cytoplasm. Tetrahydrofolates (THF)3 are present in cells as a family of metabolic cofactors that carry and chemically activate single carbons for a network of biosynthetic pathways referred to as folate-mediated one-carbon metabolism ( Fig. 1) (1, 2). Folate metabolism is compartmentalized in the cytoplasm, mitochondria, and the nucleus (2-5). In the cytoplasm, folate-activated carbons are incorporated into the 2nd and 8th positions of the purine ring and are required for the conversion of uridylate to thymidylate and for the methylation of homocysteine to methionine. Methionine can be converted to a methyl donor through its adenosylation to S-adenosylmethionine (AdoMet), a required cofactor for the methylation of DNA, RNA, proteins, lipids, and numerous small molecules. Disruptions in folatemediated one-carbon metabolism, resulting from nutritional deficiencies and/or common genetic variations, impair both DNA synthesis and chromatin methylation (1). Decreased rates of thymidylate synthesis result in increased rates of uracil misincorporation into DNA, whereas decreases in cellular methylation capacity affect both histone and cytosine methylation in chromatin. These genomic alterations are associated with genome instability, altered gene expression, and increased risk for certain cancers, developmental anomalies, and vascular and neurological disorders. However, definitive molecular mechanisms underlying these pathologies have yet to be established.Folate-activated one-carbons are derived from serine, histidine, glycine, choline, and purine catabolism, although serine is the primary source of activated carbons for folate-and AdoMetdependent one-carbon transfer reactions (6) (Fig. 1). The hydroxymethyl group of serine enters the folate-activated onecarbon pool through its THF-dependent conversion to glycine and 5,10-methylene-THF in a reaction catalyzed by the ...
The extraordinarily thin alveolar type 1 (AT1) cell constitutes nearly the entire gas exchange surface and allows passive diffusion of oxygen into the blood stream. Despite such an essential role, the transcriptional network controlling AT1 cells remains unclear. Using cell-specific knockout mouse models, genomic profiling, and 3D imaging, we found that NK homeobox 2-1 (Nkx2-1) is expressed in AT1 cells and is required for the development and maintenance of AT1 cells. Without Nkx2-1, developing AT1 cells lose 3 defining features—molecular markers, expansive morphology, and cellular quiescence—leading to alveolar simplification and lethality. NKX2-1 is also cell-autonomously required for the same 3 defining features in mature AT1 cells. Intriguingly, Nkx2-1 mutant AT1 cells activate gastrointestinal (GI) genes and form dense microvilli-like structures apically. Single-cell RNA-seq supports a linear transformation of Nkx2-1 mutant AT1 cells toward a GI fate. Whole lung ChIP-seq shows NKX2-1 binding to 68% of genes that are down-regulated upon Nkx2-1 deletion, including 93% of known AT1 genes, but near-background binding to up-regulated genes. Our results place NKX2-1 at the top of the AT1 cell transcriptional hierarchy and demonstrate remarkable plasticity of an otherwise terminally differentiated cell type.
The lung microvasculature is essential for gas exchange and commonly considered homogeneous. We show that Vascular endothelial growth factor A (Vegfa) from the epithelium specifies a distinct endothelial cell (EC) population in the postnatal mouse lung. Vegfa is predominantly expressed by alveolar type 1 (AT1) cells and locally required to specify a subset of ECs. Single cell RNA-seq identified 15-20% lung ECs as transcriptionally distinct and marked by Carbonic anhydrase 4 (Car4), which are specifically lost upon epithelial Vegfa deletion. Car4 ECs, unlike bulk ECs, have extensive cellular projections and are separated from AT1 cells by a limited basement membrane without intervening pericytes. Without Car4 ECs, the alveolar space is aberrantly enlarged despite the normal appearance of myofibroblasts. Lung Car4 ECs and retina tip ECs have common and distinct transcriptional profiles. These findings support a signaling role of AT1 cells and shed light on alveologenesis.
Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18 -/-AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18 -/-mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.
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