AP-3-dependent targeting of flippase ATP8A1 to lamellar bodies suppresses activation of YAP in alveolar epithelial type 2 cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Kook S
PROVIDER: S-EPMC8157957 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Rab38 is a rat Hermansky-Pudlak syndrome gene that plays an important role in surfactant homeostasis in alveolar type II (ATII) pneumocytes. We examined Rab38 function in regulating lamellar body (LB) morphology in ATII cells. Quantitative electron microscopy revealed that LBs in ATII cells were ?77% larger in Rab38-null fawn-hooded hypertension (FHH) than control Sprague-Dawley (SD) rats. Rab38 protein expression was restricted in lung epithelial cells but was not found in primary endothelial cells. In SD ATII cells, Rab38 protein level gradually declined during 5 days in culture. Importantly, endogenous Rab38 was present in LB fractions purified from SD rat lungs, and transiently expressed enhanced green fluorescent protein (EGFP)-tagged Rab38 labeled only the limiting membranes of a subpopulation (?30%) of LBs in cultured ATII cells. This selective targeting was abolished by point mutations to EGFP-Rab38 and was not shared by Rab7 and Rab4b, which also function in the ATII cells. Using confocal microscopy, we established a method for quantitative evaluation of the enlarged LB phenotype temporally preserved in cultured FHH ATII cells. A direct causal relationship was established when the enlarged LB phenotype was reserved and then rescued by transiently reexpressed EGFP-Rab38 in cultured FHH ATII cells. This rescuing effect was associated with dynamic EGFP-Rab38 targeting to and on LB limiting membranes. We conclude that Rab38 plays an indispensible role in maintaining LB morphology and surfactant homeostasis in ATII pneumocytes.
| S-EPMC3191760 | biostudies-literature
Project description:Lung lamellar bodies, the storage organelles for lung surfactant phosphatidylcholine (PC), maintain an acidic pH that can be increased with weak bases. This study investigates the effect of a weak base, methylamine, on the pH in lamellar bodies and on the trafficking and packaging of newly synthesized PC in lamellar bodies. Methylamine increased the pH of isolated lung lamellar bodies and of lamellar bodies in intact cells. Metabolic labelling of isolated type II cells with [methyl-3H]choline showed that although methylamine (2.5-10 mM) did not alter the labelling of cellular or microsomal PC and disaturated PC, it decreased the labelling of the PC and disaturated PC in lamellar bodies. The packaging of PC in lamellar bodies (the specific activities ratio between the PC in lamellar bodies and the microsomal PC) also decreased in a time- and concentration-dependent manner. The cellular synthesis of PC or its packaging into lamellar bodies was unaltered by brefeldin A, suggesting that the Golgi was not involved in PC packaging. Although methylamine also increased surfactant secretion, the inhibition of PC packaging in lamellar bodies seems unrelated to the secretagogue effect, (1) on the basis of metabolic consequences of increased secretion and (2) because ATP, another secretagogue, did not inhibit PC packaging. Methylamine seems to inhibit PC packaging by inhibiting trafficking of PC to lipid-rich light subcellular fractions. Together our results suggest that the trafficking of surfactant PC into lamellar bodies might be sensitive to changes in the pH of lamellar bodies.
| S-EPMC1217618 | biostudies-other
Project description:Surfactant protein A (SP-A), a lung-specific glycoprotein in pulmonary surfactant, is synthesized and secreted from the alveolar type II cells. It has been shown that SP-A is a Ca(2+)-binding protein with several binding sites and that the high-affinity site(s) is located in the C-terminal region of SP-A. In the present study we isolated the proteins from bovine lung soluble fraction that bind to SP-A in a Ca(2+)-dependent manner using DEAE-Sephacel and SP-A-conjugated Sepharose 4B. At least three different protein bands with molecular masses of 24.5, 32, and 33 kDa were observed on SDS/PAGE. The main protein, with molecular mass of 32 kDa, was identified as annexin IV by the partial-amino-acid-sequence analyses and an immunoblot analysis with anti-(annexin IV) antiserum. We also found from the immunoblot analysis that the cytosolic fraction of isolated rat alveolar type II cells contains annexin IV. In addition, when rat lung cytosol was loaded on to the lung lamellar body-conjugated Sepharose 4B in the presence of Ca2+, two proteins, with molecular masses of 32 and 60 kDa on SDS/PAGE respectively, were eluted with EGTA. The 32 kDa protein was shown to be annexin IV by an immunoblot analysis with the antiserum against annexin IV. The lung annexin IV augmented the Ca(2+)-induced aggregation of the lung lamellar bodies from rats. However, the augmentation of aggregation of the lung lamellar bodies by annexin IV was attenuated when the lamellar bodies were preincubated with polyclonal anti-SP-A antibodies. SP-A bound to annexin IV under conditions where contaminated lipid was removed. These results suggest that SP-A bound to annexin IV based on protein-protein interaction, though both proteins are phospholipid-binding proteins. All these findings suggest that the interaction between SP-A and annexin IV may have some role in alveolar type II cells.
| S-EPMC1136242 | biostudies-other
Project description:The lamellar body (LB), a concentric structure loaded with surfactant proteins and phospholipids, is an organelle specific to type 2 alveolar epithelial cells (AT2). However, the origin of LBs has not been fully elucidated. We have previously reported that autophagy regulates Weibel-Palade bodies (WPBs) formation, and here we demonstrated that autophagy is involved in LB maturation, another lysosome-related organelle. We found that during development, LBs were transformed from autophagic vacuoles containing cytoplasmic contents such as glycogen. Fusion between LBs and autophagosomes was observed in wild-type neonate mice. Moreover, the markers of autophagic activity, microtubule-associated protein 1 light chain 3B (LC3B), largely co-localized on the limiting membrane of the LB. Both autophagy-related gene 7 (Atg7) global knockout and conditional Atg7 knockdown in AT2 cells in mice led to defects in LB maturation and surfactant protein B production. Additionally, changes in autophagic activity altered LB formation and surfactant protein B production. Taken together, these results suggest that autophagy plays a critical role in the regulation of LB formation during development and the maintenance of LB homeostasis during adulthood.
| S-EPMC8771840 | biostudies-literature
Project description:BackgroundLamellar bodies are lysosome-related secretory granules and store lung surfactant in alveolar type II cells. To better understand the mechanisms of surfactant secretion, we carried out proteomic analyses of lamellar bodies isolated from rat lungs.ResultsWith peptide mass fingerprinting by Matrix Assisted Laser Desorption/Ionization - Time of Flight mass spectrometry, 44 proteins were identified with high confidence. These proteins fell into diverse functional categories: surfactant-related, membrane trafficking, calcium binding, signal transduction, cell structure, ion channels, protein processing and miscellaneous. Selected proteins were verified by Western blot and immunohistochemistry.ConclusionThis proteomic profiling of lamellar bodies provides a basis for further investigations of functional roles of the identified proteins in lamellar body biogenesis and surfactant secretion.
| S-EPMC2459160 | biostudies-literature
Project description:Ventilation is dependent upon pulmonary alveoli lined by two major epithelial cell types, alveolar type-1 (AT1) and 2 (AT2) cells. AT1 cells mediate gas exchange while AT2 cells synthesize and secrete pulmonary surfactants and serve as progenitor cells which repair the alveoli. We developed transgenic mice in which YAP was activated or deleted to determine its roles in alveolar epithelial cell differentiation. Postnatal YAP activation increased epithelial cell proliferation, increased AT1 cell numbers, and caused indeterminate differentiation of subsets of alveolar cells expressing atypical genes normally restricted to airway epithelial cells. YAP deletion increased expression of genes associated with mature AT2 cells. YAP activation enhanced DNA accessibility in promoters of transcription factors and motif enrichment analysis predicted target genes associated with alveolar cell differentiation. YAP participated with KLF5, NFIB, and NKX2-1 to regulate AGER. YAP plays a central role in a transcriptional network that regulates alveolar epithelial differentiation.
| S-EPMC8383002 | biostudies-literature
Project description:BackgroundE2A gene, which encodes two basic helix-loop-helix (bHLH) transcription factors E12 and E47, has been identified as regulator of B lymphoid hematopoiesis and suppressor of lymphoma. E47 protein was found to decrease E-cadherin expression and induce epithelial-mesenchymal transition (EMT). However, the role of E2A in colorectal cancer (CRC) metastasis is still elusive.MethodsqRT-PCR and semi-qRT-PCR were performed to determine mRNA level of E2A in CRC specimens and colorectal cancer cells. RNAi was employed to downregulate E2A expression and subsequent protein level change was evaluated by immunoblot. Cell invasion and migration capacity were detected by transwell assay using cell culture inserts with or without basement membrane matrix, respectively.ResultsE2A expression was decreased in metastatic CRC tissues. Invasion and migration assays showed downregulation of E2A increased metastatic capacity of CRC cells while forced expression of E12 or E47 could offset this effect. Both E12 and E47 suppressed EMT induced by E2A downregulation. Moreover, Yes-Associated Protein (YAP) was a downstream target of E2A and suppression of YAP inhibited the pro-migration/invasion of E2A deficiency.ConclusionOur results suggest that E2A suppresses CRC cell metastasis, at least partially if not all, by inhibiting YAP expression.
| S-EPMC3879192 | biostudies-literature
Project description:BackgroundDespite the progress of advanced target therapeutic agents and immune checkpoint inhibitors, EGFR-TKI resistance is still one of the biggest obstacles in treating lung cancer. Clinical studies with autophagy inhibitors are actively underway to overcome drug resistance.MethodsWe used PC9, PC9/GR, and HCC827/GR cell lines to evaluate the activation of autophagy and EGFR-TKI resistance. Chloroquine was applied as an autophagic blocker and verteporfin was utilized as a YAP inhibitor.ResultsIn this study, we tried to reveal the effect of autophagy adaptor p62 which is accumulated by autophagy inhibitor in EGFR-TKI-resistant lung adenocarcinoma. We identified that p62 has oncogenic functions that induce cell proliferation and invasion of EGFR-TKI-resistant lung adenocarcinoma. Interestingly, we found for the first time that YAP regulates p62 transcription through ERK, and YAP inhibition can suppress the expression of oncogenic p62. We also confirmed that the expressions of p62 and YAP have a positive correlation in EGFR-mutant lung adenocarcinoma patients. To block cell survival via perturbing YAP-p62 axis, we treated EGFR-TKI-resistant lung cancer cells with YAP inhibitor verteporfin. Remarkably, verteporfin effectively caused the death of EGFR-TKI-resistant lung cancer cells by decreasing the expressions of p62 with oncogenic function, YAP, and its target PD-L1. So, the cumulative effect of oncogenic p62 should be considered when using autophagy inhibitors, especially drugs that act at the last stage of autophagy such as chloroquine and bafilomycin A1.ConclusionFinally, we suggest that targeting YAP-p62 signaling axis can be useful to suppress the EGFR-TKI-resistant lung cancer. Therefore, drug repurposing of verteporfin for lung cancer treatment may be valuable to consider because it can inhibit critical targets: p62, YAP, and PD-L1 at the same time.
| S-EPMC7926029 | biostudies-literature
Project description:Mevastatin (MVS) has been previously shown to induce heme oxygenase (HO)-1 expression through Nox/ROS-dependent PDGFR?/PI3K/Akt/Nrf2/ARE axis in human pulmonary alveolar epithelial cells (HPAEpiCs). However, alternative signaling pathways might involve in MVS-induced HO-1 expression. We found that tumor necrosis factor ? (TNF?) induced vascular cell adhesion protein 1 (VCAM-1) expression and NF-?B p65 phosphorylation which were attenuated by pretreatment with MVS via up-regulation of HO-1, determined by Western blot and real-time qPCR. TNF?-induced VCAM-1 expression was attenuated by an NF-?B inhibitor, Bay117082. The inhibitory effects of MVS were reversed by tin protoporphyrin (SnPP)IX (an inhibitor of HO-1 activity). In addition, pretreatment with the inhibitor of pan-Protein kinase C (PKC) (GF109203X), PKC? (Gö6983), Pyk2 (PF431396), p38? MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA), and transfection with their respective siRNAs abolished MVS-induced HO-1 expression in HPAEpiCs. c-Jun (one of AP-1 subunits) was activated by PKC?, Pyk2, p38? MAPK, and JNK1/2, which turned on the transcription of the homx1 gene. The interaction between c-Jun and HO-1 promoter was confirmed by a chromatin immunoprecipitation (ChIP) assay, which was attenuated by these pharmacological inhibitors. These results suggested that MVS induces AP-1/HO-1 expression via PKC?/Pyk2/p38? MAPK- or JNK1/2-dependent c-Jun activation, which further binds with AP-1-binding site on HO-1 promoter and suppresses the TNF?-mediated inflammatory responses in HPAEpiCs. Thus, upregulation of the AP-1/HO-1 system by MVS exerts a potentially therapeutic strategy to protect against pulmonary inflammation.
| S-EPMC7175369 | biostudies-literature
Project description:CCCTC-binding factor (CTCF) has a key role in higher-order chromatin architecture that is important for establishing and maintaining cell identity by controlling gene expression. In the mature cerebellum, CTCF is highly expressed in Purkinje cells (PCs) as compared with other cerebellar neurons. The cerebellum plays an important role in motor function by regulating PCs, which are the sole output neurons, and defects in PCs cause motor dysfunction. However, the role of CTCF in PCs has not yet been explored. Here we found that the absence of CTCF in mouse PCs led to progressive motor dysfunction and abnormal dendritic morphology in those cells, which included dendritic self-avoidance defects and a proximal shift in the climbing fibre innervation territory on PC dendrites. Furthermore, we found the peculiar lamellar structures known as "giant lamellar bodies" (GLBs), which have been reported in PCs of patients with Werdnig-Hoffman disease, 13q deletion syndrome, and Krabbe disease. GLBs are localized to PC dendrites and are assumed to be associated with neurodegeneration. They have been noted, however, only in case reports following autopsy, and reports of their existence have been very limited. Here we show that GLBs were reproducibly formed in PC dendrites of a mouse model in which CTCF was deleted. GLBs were not noted in PC dendrites at infancy but instead developed over time. In conjunction with GLB development in PC dendrites, the endoplasmic reticulum was almost absent around the nuclei, the mitochondria were markedly swollen and their cristae had decreased drastically, and almost all PCs eventually disappeared as severe motor deficits manifested. Our results revealed the important role of CTCF during normal development and in maintaining PCs and provide new insights into the molecular mechanism of GLB formation during neurodegenerative disease.
| S-EPMC9706876 | biostudies-literature