Knockdown of flotillin-2 inhibits lung surfactant secretion by alveolar type II cells.
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ABSTRACT:
SUBMITTER: Chintagari NR
PROVIDER: S-EPMC2430057 | biostudies-literature | 2008 Jun
REPOSITORIES: biostudies-literature
Publications
Knockdown of flotillin-2 inhibits lung surfactant secretion by alveolar type II cells.
Chintagari Narendranath Reddy NR Gou Deming D Liu Lin L
Cell research 20080601 6
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Project description:Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase) is the enzyme responsible for pumping H(+) into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase) dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1), an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+) chelator, BAPTA-AM, the protein kinase C (PKC) inhibitor, staurosporine, and the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), KN-62. Baf A1 induced Ca(2+) release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+) pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+) mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.
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Project description:MicroRNA (miRNA) critically controls gene expression in many biological processes, including lung growth and pulmonary surfactant biosynthesis. The present study was conducted to investigate whether miR-20a-5p had such regulatory functions on alveolar type II (AT-II) cells. To accomplish this, miR-20a-5p-overexpressed and miR-20a-5p-inhibited adenoviral vectors were constructed and transfected into cultured AT-II cells that were isolated from rat foetal lungs of 19 days' gestation. Transfection efficiency was confirmed by observing the fluorescence of green fluorescent protein (GFP) carried by the viral vector, whereas miR-20a-5p levels were verified by real-time PCR. The CCK-8 assay was used to compare the proliferation ability of AT-II cells that had over- or underexpressed miR-20a-5p. The expression of surfactant-associated proteins (SPs) and phosphatase and tensin homolog (PTEN) was measured by real-time PCR and Western blotting. In AT-II cells, transfection resulted in over- or under-regulation of miR-20a-5p. While overexpression of miR-20a-5p promoted pulmonary surfactant gene expression, its underexpression inhibited it. Consistent with its role in negatively regulating the pulmonary surfactant gene, an opposite pattern was observed for miR-20a-5p regulation of PTEN. As a result, when miR-20a-5p was rendered overexpressed, PTEN was down-regulated. By contrast, when miR-20a-5p was underexpressed, PTEN was up-regulated. Neither overexpression nor underexpression of miR-20a-5p altered the cell proliferation. miR-20a-5p plays no role in proliferation of foetal AT-II cells but is a critical regulator of surfactant gene expression. The latter appears to be achieved through a regulatory process that implicates expression of PTEN.
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Project description:ErbB4 receptor and thyroid transcription factor (TTF)-1 are important modulators of fetal alveolar type II (ATII) cell development and injury. ErbB4 is an upstream regulator of TTF-1, promoting its expression in MLE-12 cells, an ATII cell line. Both proteins are known to promote surfactant protein-B gene (SftpB) and protein (SP-B) expression, but their feedback interactions on each other are not known. We hypothesized that TTF-1 expression has a feedback effect on ErbB4 expression in an in-vitro model of isolated mouse ATII cells. We tested this hypothesis by analyzing the effects of overexpressing HER4 and Nkx2.1, the genes of ErbB4 and TTF-1 on TTF-1 and ErbB4 protein expression, respectively, as well as SP-B protein expression in primary fetal mouse lung ATII cells. Transient ErbB4 protein overexpression upregulated TTF-1 protein expression in primary fetal ATII cells, similarly to results previously shown in MLE-12 cells. Transient TTF-1 protein overexpression down regulated ErbB4 protein expression in both cell types. TTF-1 protein was upregulated in primary transgenic ErbB4-depleted adult ATII cells, however SP-B protein expression in these adult transgenic ATII cells was not affected by the absence of ErbB4. The observation that TTF-1 is upregulated in fetal ATII cells by ErbB4 overexpression and also in ErbB4-deleted adult ATII cells suggests additional factors interact with ErbB4 to regulate TTF-1 levels. We conclude that the interdependency of TTF-1 and ErbB4 is important for surfactant protein levels. The interactive regulation of ErbB4 and TTF-1 needs further elucidation.
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Project description:Surfactant proteins (SPs) are important lipoprotein complex components, expressed in alveolar epithelial cells type II (AEC-II), and playing an essential role in maintenance of alveolar integrity and host defence. Because expressions of SPs are regulated by cyclic adenosine monophosphate (cAMP), we hypothesized that phosphodiesterase (PDE) inhibitors, influence SP expression and release. Analysis of PDE activity of our AEC-II preparations revealed that PDE4 is the major cAMP hydrolysing PDE in human adult AEC-II. Thus, freshly isolated human AEC-II were stimulated with two different concentrations of the PDE4 inhibitor roflumilast-N-oxide (3 nM and 1 µM) to investigate the effect on SP expression. SP mRNA levels disclosed a large inter-individual variation. Therefore, the experiments were grouped by the basal SP expression in low and high expressing donors. AEC-II stimulated with Roflumilast-N-oxide showed a minor increase in SP-A1, SP-C and SP-D mRNA mainly in low expressing preparations. To overcome the effects of different basal levels of intracellular cAMP, cyclooxygenase was blocked by indomethacin and cAMP production was reconstituted by prostaglandin E2 (PGE2). Under these conditions SP-A1, SP-A2, SP-B and SP-D are increased by roflumilast-N-oxide in low expressing preparations. Roflumilast-N-oxide fosters the expression of SPs in human AEC-II via increase of intracellular cAMP levels potentially contributing to improved alveolar host defence and enhanced resolution of inflammation.
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