Jejunal gene expression in surfactant protein D (Sftpd) knockout and wild-type mice after doxorubicin-induced intestinal toxicity
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE335354 | GEO | 2026/06/24
REPOSITORIES: GEO
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Project description:This study uses microarray analyses to examine gene expression profiles for Mycobacterium tuberculosis (Mtb) induced by exposure in vitro to bovine lung surfactant preparations that vary in apoprotein content: (i) whole lung surfactant (WLS) containing the complete mix of endogenous lipids and surfactant proteins (SP)-A, -B, -C, and -D; (ii) extracted lung surfactant (CLSE) containing lipids plus SP-B and -C; (iii) column-purified surfactant lipids (PPL) containing no apoproteins, and (iv) purified human SP-A. Exposure to WLS evoked a multitude of transcriptional responses in Mtb, with 52 genes up-regulated and 23 genes down-regulated at 30 min exposure, plus 146 genes up-regulated and 27 genes down-regulated at 2 h. Notably, WLS rapidly induced several membrane-associated lipases that presumptively act on surfactant lipids as substrates, and a large number of genes involved in the synthesis of phthiocerol dimycocerosate (PDIM), a cell wall component known to be important in macrophage interactions and Mtb virulence. Exposure of Mtb to CLSE, PPL, or purified SP-A caused a substantially weaker transcriptional response (≤ 20 genes were induced) suggesting that interactions among multiple lipid-protein components of WLS may contribute to its effects on Mtb transcription.
2009-06-01 | GSE14005 | GEO
Project description:This study uses microarray analyses to examine gene expression profiles for Mycobacterium tuberculosis (Mtb) induced by exposure in vitro to bovine lung surfactant preparations that vary in apoprotein content: (i) whole lung surfactant (WLS) containing the complete mix of endogenous lipids and surfactant proteins (SP)-A, -B, -C, and -D; (ii) extracted lung surfactant (CLSE) containing lipids plus SP-B and -C; (iii) column-purified surfactant lipids (PPL) containing no apoproteins, and (iv) purified human SP-A. Exposure to WLS evoked a multitude of transcriptional responses in Mtb, with 52 genes up-regulated and 23 genes down-regulated at 30 min exposure, plus 146 genes up-regulated and 27 genes down-regulated at 2 h. Notably, WLS rapidly induced several membrane-associated lipases that presumptively act on surfactant lipids as substrates, and a large number of genes involved in the synthesis of phthiocerol dimycocerosate (PDIM), a cell wall component known to be important in macrophage interactions and Mtb virulence. Exposure of Mtb to CLSE, PPL, or purified SP-A caused a substantially weaker transcriptional response (⤠20 genes were induced) suggesting that interactions among multiple lipid-protein components of WLS may contribute to its effects on Mtb transcription. Whole lung surfactant (WLS) was obtained by centrifugation of lavage fluid (0.15M NaCl) from the intact lungs of freshly killed calves (ONY, Inc, Amherst, NY). Centrifugation was done at 12,000 x g at room temperature to pellet the active large aggregate surfactant fraction. CLSE was prepared by extraction of WLS into chloroform-methanol by the method of Bligh and Dyer (1959), as described in our prior work (Notter et al. 2002). CLSE is conceptually identical to the substance of the clinical surfactant Infasurf®. The composition of CLSE by weight was approximately 94% phospholipid, 4.5% cholesterol, and 1.5% protein (a mixture of SP-B and SP-C) as defined previously (Notter et al. 2002). Mixed lung surfactant phospholipids (PPL) were isolated from CLSE by column chromatography with an elution solvent of 1:1 (v/v) chloroform-methanol plus 5% 0.1 N HCl. Two passes through a Sephadex LH-20 column (Pharmacia-LKB Biotechnology, Piscataway, NJ) separated phospholipids away from hydrophobic surfactant apoproteins and neutral lipids. Acid remaining in PPL fractions was removed by a second chloroform-methanol extraction. Final PPL had a protein content below the limits of detection of both the Folin-phenol assay of Lowry et al. (1951) and the amido black assay of Kaplin and Pedersen (1989) modified by the addition of 15% sodium dodecyl sulfate (SDS) to allow accurate quantitation in the presence of lipid. Human SP-A was a gift from Dr. J. R. Wright at Duke University, and was isolated from bronchoalveolar lavage from patients with alveolar proteinosis as described previously (Wright et al. 1987). For exposure studies with Mtb, amounts of WLS, CLSE, and PPL used per experiment were 3.3 mg in a total volume of 4 ml. In preliminary studies, this concentration was shown to induce a transcriptional response in Mtb without affecting viability after a 2 h treatment as determined by serial dilutions compared to untreated Mtb. SP-A was studied at a level of 0.165 mg based on its putative concentration of approximately 5% in endogenous surfactant. To determine the effects of lung surfactant on the transcriptional response of Mtb, preparations of WLS, CLSE, or PPL (in 650 µl PBS) were initially coated onto the bottom surface of a 25 cm2 cell culture flask for 30 min at 37oC to mimic a surfactant layer. A minimum of two independent biological replicates were studied for each surfactant preparation. Purified human SP-A was also examined for its effects on Mtb transcription, and phosphate buffered saline (PBS) was used as a âno treatmentâ control. In experiments with SP-A (in 650 µl PBS), the pure apoprotein was either pre-coated on the flask as done with the surfactant preparations, or was directly added and incubated with Mtb during study. Mtb was added to the flask in 3.35 ml of DMEM (supplemented with 2% FCS and 5 mM HEPES) and exposed to coated surfactant/SP-A or PBS control for 30 min or 2h at 37oC, followed by RNA isolation as detailed by Rohde et al. (2008). GTC lysis/stabilization buffer was added, and pelleted Mtb were washed and then digested with lysozyme. TRIzol (Invitrogen) was added, and organisms were disrupted using glass beads in a Mini-Bead Beater (BioSpec Products, Inc. Bartlesville, OK). After extraction of Mtb lysates with chloroform, ethanol was added to the aqueous phase before loading samples onto RNeasy mini columns (Qiagen, Valencia, CA). Subsequent steps were then performed according to the manufacturerâs instructions. Following elution with RNase-free water, the RNA samples were treated with DNase (Turbo DNAfree, Applied Biosystems, Foster City, CA), and stored at -70oC until analysis. The MessageAmpTMII-Bacteria system (Applied Biosystems) was used to amplify mycobacterial RNA for all samples [16]. Total RNA was reverse transcribed to yield double-stranded amino-allyl modified aRNA, which was labeled with Alexa Fluor 555 and Alexa Fluor 647 (Invitrogen). Excess reactive dyes were removed with a Mega Clear purification kit (Applied Biosystems). Next, Alexa-labeled aRNA from paired samples was dried using a Speedvac, and re-suspended in hybridization buffer (5x SSC, 25% formamide, 0.15% SDS, and 25 μg salmon sperm DNA). The mixed samples were denatured by being heated to 95oC for and then cooled to 50oC prior to loading them onto microarrays. The arrays were hybridized for approximately 15 h at 45oC, washed and then dried by centrifugation. Fluorescence intensity data from each array were collected with a GenePix 4000B scanner (Axon Instruments, Union City, CA) using GenePixPro3.0 software (Axon Instruments). Image analysis, i.e. spot identification, background measurements, quality assessment and flagging were conducted with Imagene software (Version 6.0, BioDiscovery, Inc., El Segundo, CA). Poor quality spots were filtered and removed from the analysis. Subsequent normalization, statistical analysis (i.e. t-test), and visualization of array data was conducted using Genespring (Version 7, Agilent Technologies, Palo Alto, CA).
2010-05-18 | E-GEOD-14005 | biostudies-arrayexpress
Project description:Mutations in the SFTPC gene associated with interstitial lung disease in human patients result in misfolding, endoplasmic reticulum (ER) retention, and degradation of the encoded surfactant protein C (SP-C) proprotein. To identify candidate genes involved in ER quality control of SP-C, HEK293 cells were transiently transfected with mutant SP-C (SP-CΔexon4 or SP-CL188Q), SP-CWT, or vector cDNAs, and global changes in gene expression were assessed by microarray analyses. Microarray analysis demonstrated that the SPC exon 4 deletion and SPC L188Q mutations invoke very similar transcriptional profiles including the activation of major players mediating ER response and unfolding protein response (UPR) in transient transfection system. In combination with promoter scan (UPRE, ERSE, XBP1 sites) and protein domain analysis (Finding ER Lumen, ER Membrane retention signal, J-Domain and Leucine Zipper domain), we were able to not only verify the known ERAD components (XBP1, Bip, Erdj4&5), but also identify multiple ER components which may play critical roles in the detection and /or degradation of mutant SPC, which in turn will help us to gain better understanding of the entire mammalian ERAD machinery. samples consisted of HEK293 cells transiently transfected with wild-type SP-C (WT), the delta exon4 mutant of SP-C (Ex4) or L188Q mutant of SP-C (LQ). Groups were performed in triplicate.
2009-05-14 | E-GEOD-16054 | biostudies-arrayexpress
Project description:Mutations in the SFTPC gene associated with interstitial lung disease in human patients result in misfolding, endoplasmic reticulum (ER) retention, and degradation of the encoded surfactant protein C (SP-C) proprotein. To identify candidate genes involved in ER quality control of SP-C, HEK293 cells were transiently transfected with mutant SP-C (SP-CΔexon4 or SP-CL188Q), SP-CWT, or vector cDNAs, and global changes in gene expression were assessed by microarray analyses. Microarray analysis demonstrated that the SPC exon 4 deletion and SPC L188Q mutations invoke very similar transcriptional profiles including the activation of major players mediating ER response and unfolding protein response (UPR) in transient transfection system. In combination with promoter scan (UPRE, ERSE, XBP1 sites) and protein domain analysis (Finding ER Lumen, ER Membrane retention signal, J-Domain and Leucine Zipper domain), we were able to not only verify the known ERAD components (XBP1, Bip, Erdj4&5), but also identify multiple ER components which may play critical roles in the detection and /or degradation of mutant SPC, which in turn will help us to gain better understanding of the entire mammalian ERAD machinery.
2009-05-14 | GSE16054 | GEO
Project description:This project has the goal of determine the impact of the missense mutation I73T on the surfactant protein SP-C and the role of EMC3 in regulating the trafficking of this protein.
2024-10-30 | PXD029962 | Pride
Project description:Mutations in the gene encoding surfactant protein C (SFTPC) have been linked to interstitial lung disease in children and adults. Expression of the index mutation, SP-Cdeltaexon4, in transiently transfected cells and type II cells of transgenic mice resulted in misfolding of the proprotein, activation of ER stress pathways and cytotoxicity. In the current study we show that stably transfected cells adapted to chronic ER stress imposed by constitutive expression of SP-Cdeltaexon4 via an NF-kB-dependent pathway. However, infection of cells expressing SP-Cdeltaexon4 with respiratory syncytial virus resulted in significantly enhanced cytotoxicity associated with accumulation of the mutant proprotein, pronounced activation of the unfolded protein response and cell death. Adaptation to chronic ER stress imposed by misfolded SP-C was associated with increased susceptibility to viral-induced cell death. The wide variability in the age of onset of ILD in patients with SFTPC mutations may be related to exposure to an environmental insult that ultimately overwhelms the homeostatic, cytoprotective response. Keywords: transcriptional response to misfolded protein due to genetic modification
2006-03-20 | GSE2980 | GEO
Project description:The amount of pulmonary surfactant within type II cells and in the alveolar space, referred to as surfactant pool sizes, are tightly regulated. The molecular pathways that sense and regulate surfactant pool size within the alveolus have not been identified and constitute a fundamental knowledge gap in the field. Our data show that mice with a germline mutation in the orphan G-protein-coupled receptor, GPR116, have a 30-fold accumulation of surfactant phospholipids that causes respiratory distress in adult animals. This phenotype is associated with increased surfactant secretion and induction of the purinergic receptor P2RY2 in young animals, and lipid-laden macrophages and alveolar destruction in older animals. We further demonstrate that GPR116 mRNA expression is developmentally regulated in the murine lung with peak expression at birth when surfactant pool sizes are maximal. Within the lung, GPR116 protein expression is restricted to the apical plasma membrane of alveolar type I and type II epithelial cells. To better understand the roles and molecular mechanisms by which Gpr116 influences gene expression in lung, the effect of cell-selective deletion of Gpr116 (Gpr116D/D) on genome-wide mRNA expression profiles was determined in murine type II alveolar epithelial cells. Differentially expressed genes were identified from Affymetrix Murine GeneChips analysis and subjected to gene ontology classification promoter analysis, pathway mapping and literature mining.
2013-04-10 | E-GEOD-41417 | biostudies-arrayexpress
Project description:The amount of pulmonary surfactant within type II cells and in the alveolar space, referred to as surfactant pool sizes, are tightly regulated. The molecular pathways that sense and regulate surfactant pool size within the alveolus have not been identified and constitute a fundamental knowledge gap in the field. Our data show that mice with a germline mutation in the orphan G-protein-coupled receptor, GPR116, have a 30-fold accumulation of surfactant phospholipids that causes respiratory distress in adult animals. This phenotype is associated with increased surfactant secretion and induction of the purinergic receptor P2RY2 in young animals, and lipid-laden macrophages and alveolar destruction in older animals. We further demonstrate that GPR116 mRNA expression is developmentally regulated in the murine lung with peak expression at birth when surfactant pool sizes are maximal. Within the lung, GPR116 protein expression is restricted to the apical plasma membrane of alveolar type I and type II epithelial cells.
2013-04-10 | GSE41417 | GEO
Project description:Pulmonary surfactant protein D (SP-D) is an endogenous innate immune factor known to be involved in several immune related diseases and has also been linked to lung adenocarcinoma cell growth. By analyzing single-cell, bulk, and spatial transcriptomics of human clinical samples, we have demonstrated significantly reduced expression in lung cancer cells compared to healthy cells, along with a distinct localization of SP-D expression in stromal areas adjacent to the tumor cells and at the periphery. Interestingly, a near-complete loss of SP-D expression was observed in metastatic tissue, suggesting its association with a more aggressive and metastatic phenotype. In this context, we show that SP-D re-expression in A549 adenocarcinoma cell line significantly decreased tumor growth and spontaneous metastasis formation in vivo and furthermore, that intranasal administration of recombinant SP-D signicantly reduced lung tumor burden. Mechanistically, we demonstrated that SP-D supresses interleukin 4 (IL4)/IL13 signaling pathways. Intriguingly, our data revealed that recombinant SP-D binds directly to the IL4 receptor, leading to the effective inhibition of the IL4 signaling pathway. Collectively, these findings demonstrate that SP-D acts as a tumor supressor inhibiting both tumor growth and metastatic progression potentially through blokage of the IL4/STAT6 signaling pathway. This provides a rationale of SP-D as a promising therapeutic agent for modulating crucial signaling pathways involved in cancer development and progression.
2025-12-31 | GSE249805 | GEO
Project description:Covid-19 syndrome is characterized by an acute lung injury phenotype marked by hypoxemic respiratory failure and high mortality. Alveolar Type 2 (AT2) cells are essential for gas exchange, repair, and regeneration of distal lung epithelium. We have shown that the causative agent, SARS-CoV-2 and other members of the b coronavirus genus induces an ER stress response in vitro however the consequences for host AT2 cell function in vivo are less understood. To study this, two murine models of coronavirus infection were employed– mouse hepatitis virus-1 (MHV-1) in A/J mice and a C57BL6/j adapted SARS-CoV-2 strain. MHV-1 infected mice exhibited dose-dependent weight loss through Day 8 (8dpi) with histological evidence of distal lung injury accompanied by elevated BALF cell counts and total protein. AT2 cells isolated at 4dpi and 8dpi showed evidence of both viral infection and increases in Bip /GRP78 expression, consistent with activation of the unfolded protein response (UPR). AT2 UPR signaling included elevations in spliced XBP-1 indicating activation of IRE1a signaling as well as a biphasic response in PERK signaling mark by phosphorylated eIF2a protein levels and expression of Atf4, Ppp1r15a, and Ddit3. Viral UPR activation was accompanied by marked reductions in AT2 and BALF surfactant protein (SP-B, SP-C) content, increases in surfactant surface tension, and emergence of a re-programmed epithelial cell population (Krt8+, Cldn4+, and Gdf15+) which was attenuated by treatment with the IRE1a inhibitor OPK711. As proof-of-concept, C57BL6 mice infected with mouse-adapted SARS-CoV-2 demonstrated similar lung injury and evidence of disrupted surfactant homeostasis. We conclude that hypoxic respiratory failure from b-coronavirus infection results from an aberrant AT2 cell host response activating multiple ER stress pathways, altering surfactant metabolism / function, and changing AT2 endophenotypes offering a mechanistic link between SARS-CoV-2 infection, AT2 cell biology, and hypoxemic respiratory failure.
2024-06-27 | GSE244820 | GEO