Kathrin Barth

Caveolin-1 influences P2X7 receptor expression and localization in mouse lung alveolar epithelial cells.

The P2X7 receptor has recently been described as a marker for lung alveolar epithelial type I cells. Here, we demonstrate both the expression of P2X7 protein and its partition into lipid rafts in the mouse lung alveolar epithelial cell line E10. A significant degree of colocalization was observed between P2X7 and the raft marker protein Caveolin‐1; also, P2X7 protein was associated with caveolae. A marked reduction in P2X7 immunoreactivity was observed in lung sections prepared from Caveolin‐1‐knockout mice, indicating that Caveolin‐1 expression was required for full expression of P2X7 protein. Indeed, suppression of Caveolin‐1 protein expression in E10 cells using short hairpin RNAs resulted in a large reduction in P2X7 protein expression. Our data demonstrate a potential interaction between P2X7 protein and Caveolin‐1 in lipid rafts, and provide a basis for further functional and biochemical studies to probe the physiologic significance of this interaction.

Bleomycin and its role in inducing apoptosis and senescence in lung cells - modulating effects of caveolin-1.

Bleomycin, a widely used anti-tumor agent, is well-known to cause single- and double-strand breaks in cellular DNA in vivo and in vitro leading finally to genomic instability of damaged cells. Bleomycin causes an increase of reactive oxygen species resulting in oxidative stress and pulmonary fibrosis. Further, bleomycin induces apoptosis and senescence in epithelial and non-epithelial cells of the lung. Caveolin-1 is a scaffold protein of caveolae, which are particularly abundant in alveolar epithelial type I cells, in endothelial and smooth muscle cells, and in fibroblasts of lung tissue. Caveolin-1 directly interacts with signaling molecules and effects diverse signaling pathways regulating cell proliferation, apoptosis, differentiation and growth. In this review we discuss aspects of bleomycin resistance. We summarize recent data about the effects of bleomycin in terms of lung cell biology and emphasize that bleomycin-induced injury of lung cells is accompanied by altered expression levels of caveolin-1. Caveolin-1 is involved in bleomycin-induced apoptosis and senescence of normal and lung cancer cells. Investigating the role of caveolin-1 may provide new tools for therapeutic interventions in lung disease and for the understanding of tumor biology.

Immunocytochemical and biochemical detection of EMMPRIN in the rat tooth germ: differentiation-dependent co-expression with MMPs and co-localization with caveolin-1 in membrane rafts of dental epithelial cells.

In tooth development matrix metalloproteinases (MMPs) are under the control of several regulatory mechanisms including the upregulation of expression by inducers and downregulation by inhibitors. The aim of the present study was to monitor the occurrence and distribution pattern of the extracellular matrix metalloproteinase inducer (EMMPRIN), the metalloproteinases MMP-2 and MT1-MMP and caveolin-1 during the cap and bell stage of rat molar tooth germs by means of immunocytochemistry. Strong EMMPRIN immunoreactivity was detected on the cell membranes of ameloblasts and cells of the stratum intermedium in the bell stage of the enamel organ. Differentiating odontoblasts exhibited intense EMMPRIN immunoreactivity, especially at their distal ends. Caveolin-1 immunoreactivity was evident in cells of the internal enamel epithelium and in ameloblasts. Double immunofluorescence studies revealed a focal co-localization between caveolin-1 and EMMPRIN in ameloblastic cells. Finally, western blotting experiments demonstrated the expression of EMMPRIN and caveolin-1 in dental epithelial cells (HAT-7 cells). A substantial part of EMMPRIN was detected in the detergent-insoluble caveolin-1-containing low-density raft membrane fraction of HAT-7 cells suggesting a partial localization within lipid rafts. The differentiation-dependent co-expression of MMPs with EMMPRIN in the enamel organ and in odontoblasts indicates that EMMPRIN takes part in the induction of proteolytic enzymes in the rat tooth germ. The localization of EMMPRIN in membrane rafts provides a basis for further investigations on the role of caveolin-1 in EMMPRIN-mediated signal transduction cascades in ameloblasts.

Characterization of the molecular interaction between caveolin-1 and the P2X receptors 4 and 7 in E10 mouse lung alveolar epithelial cells

P2X(4) and P2X(7) receptors are abundantly expressed in alveolar epithelial cells, and are thought to play a role in regulating fluid haemostasis. Here, we analyzed the expression and localization of the P2X(4)R, and characterized the interaction between Cav-1 and both P2X(4)R and P2X(7)R in the mouse alveolar epithelial cell line E10. Using the biotinylation assay, we found that only glycosylated P2X(4)R is exposed at the cell surface. Triton X-100 solubility experiments and sucrose gradient centrifugation revealed that P2X(4)R was partially localized in Cav-1 rich membrane fractions. Cholesterol depletion with Mbeta-CD displaced Cav-1 and P2X(4)R from the low-density to the high-density fractions. Suppression of Cav-1 protein expression using short hairpin RNAs resulted in a large reduction in P2X(4)R levels. Double immunofluorescence showed that P2X(4)R and Cav-1 partially colocalize in vitro. Using the GST pull-down assay, we showed that Cav-1 interacts in vitro with both P2X(4)R and P2X(7)R. Co-immunoprecipitation experiments confirmed the interaction between P2X(7)R and Cav-1. ATP stimulation increased the level of P2X(4)R in the lipid raft/caveolae fraction, whereas Cav-1 content remained constant. Our results support recent evidence that P2X receptors are present in both raft and non-raft compartments of the plasma membrane and thus exhibit variable ATP sensitivity.

Epithelial vs myofibroblast differentiation in immortal rat lung cell lines—modulating effects of bleomycin

Two alveolar epithelial cell lines R3/1 and L2 were screened by immunocytochemical and RT-PCR analysis of epithelial and mesenchymal/contractile marker proteins. R3/1 and L2 cells were tested for their sensitivity to bleomycin (BLM), an anticancer drug, which is proposed to induce changes in lung cell differentiation. Both epithelial cell lines exhibited a mixed phenotype consisting of epithelial (E-cadherin, aquaporin-5 and cytokeratin 8) and myofibroblast-like (vimentin, α-SMA and caveolin-3) properties suggesting that the cell lines are arrested in vitro at a certain developmental stage during epithelial–mesenchymal transition (EMT). BLM treatment of R3/1 cells resulted in a partial reversal of this process modifying the cells in an epithelial direction, e.g., upregulation of E-cadherin, aquaporin-5 and other lung epithelial antigens at the mRNA and protein level. L2 cells showed similar alterations following BLM exposure.Immunohistochemical investigation of lung tissue from two different animal models of BLM-induced fibrosis (mouse and rat), revealed no signs of EMT, e.g., myofibroblastic differentiation of alveolar epithelial cells in situ. Immunohistological analysis of tissue samples of the rat model showed a heterogeneous population of myofibroblasts (α-SMA+/caveolin-3+, α-SMA-/caveolin-3+, and α-SMA+/caveolin-3−). These results suggest that BLM, on one hand, induces fibrosis and on the other hand possibly suppresses EMT during fibrogenesis.

Membrane compartments and purinergic signalling: occurrence and function of P2X receptors in lung

P2X receptors are cation‐selective ion channels activated by extracellular ATP. They form homo‐ and heterotrimeric complexes that differ in their functional properties and subcellular localization. These membrane ion channels are also expressed in pulmonary epithelial cells. Recent work indicates that alveolar epithelial type I cells selectively express P2X4 and P2X7 receptor subtypes in addition to a large number of other ion channels present in the alveolar epithelium. Up‐ or downregulation of their expression is associated with several disease states. This minireview analyses the role of P2X receptors and of extracellular ATP and adenosine in lung disease, the relationship of P2X receptors to other ion channels in the alveolar epithelium and their distribution in lipid rafts/caveolae.

Saccharomyces cerevisiae translational activator Cbs2p is associated with mitochondrial ribosomes

A characteristic feature of the mitochondrial expression system in Saccharomyces cerevisiae is the requirement for gene-specific translational activator proteins. Translation of mitochondrial apocytochrome b mRNA requires the nucleus-encoded proteins Cbs1p and Cbs2p. These proteins are thought to tether cytochrome b mRNA to the mitochondrial inner membrane via binding to the 5′ untranslated mRNA leader. Here, we demonstrate by the use of affinity chromatography and coimmunoprecipitation that Cbs2p interacts with the mitoribosomes. We further provide evidence that the C-terminus of Cbs2p is important for ribosome association, while the N-terminal portion is essential for the formation of homomeric structures.

A Co-Culture System with an Organotypic Lung Slice and an Immortal Alveolar Macrophage Cell Line to Quantify Silica-Induced Inflammation

There is growing evidence that amorphous silica nanoparticles cause toxic effects on lung cells in vivo as well as in vitro and induce inflammatory processes. The phagocytosis of silica by alveolar macrophages potentiates these effects. To understand the underlying molecular mechanisms of silica toxicity, we applied a co-culture system including the immortal alveolar epithelial mouse cell line E10 and the macrophage cell line AMJ2-C11. In parallel we exposed precision-cut lung slices (lacking any blood cells as well as residual alveolar macrophages) of wild type and P2rx7−/− mice with or without AMJ2-C11 cells to silica nanoparticles. Exposure of E10 cells as well as slices of wild type mice resulted in an increase of typical alveolar epithelial type 1 cell proteins like T1α, caveolin-1 and -2 and PKC-β1, whereas the co-culture with AMJ2-C11 showed mostly a slightly lesser increase of these proteins. In P2rx7−/− mice most of these proteins were slightly decreased. ELISA analysis of the supernatant of wild type and P2rx7−/− mice precision-cut lung slices showed decreased amounts of IL-6 and TNF-α when incubated with nano-silica. Our findings indicate that alveolar macrophages influence the early inflammation of the lung and also that cell damaging reagents e.g. silica have a smaller impact on P2rx7−/− mice than on wild type mice. The co-culture system with an organotypic lung slice is a useful tool to study the role of alveolar macrophages during lung injury at the organoid level.

Activation of P2X7R and downstream effects in bleomycin treated lung epithelial cells

Changes in intracellular calcium concentration [Ca(2+)](i) are believed to influence the proliferation and differentiation of airway epithelial cells both in vivo and in vitro. In the present study, using mouse alveolar epithelial E10 cells, we demonstrated that the treatment of lung epithelial cells with BLM resulted in elevated intracellular Ca(2+) levels. BLM further increased P2rx7 mRNA expression and P2X7R protein levels, paralleled by increased PKC-β1 levels. BLM treatment or stimulation of the P2X7R with the P2X7R agonist BzATP induced translocation of PKC-β1 from the cytoplasm to the membrane. The expression of PKC-β1 was repressed by the P2X7R inhibitor oxATP, suggesting that PKC-β1 is downstream of P2X7R activation. Furthermore, cells exposed to BLM contained increased amounts of P2X7R and PKC-β1 in Cav-1 containing lipid raft fractions. The comparison of lung tissues from wild-type and P2rx7(-/-) mice revealed decreased protein and mRNA levels of PKC-β1 and CaM as well as decreased immunoreactivity for PKC-β1. The knockdown of P2X7R in alveolar epithelial cells resulted also in a loss of PKC-β1. These data suggest that the effect of P2X7R on expression of PKC-β1 detected in alveolar epithelial cells is also functioning in the animal model. Immunohistochemical evaluation of fibrotic lungs derived from a BLM-induced mouse model revealed a strong increase in PKC-β1 immunoreactivity. The present experiments demonstrated that the increased expression of P2X7R influences PKC-β1. We predict that increased Ca(2+) concentration stimulates PKC-β1, whereas the prerequisite for activating PKC-β1 after P2X7R increase remained to be determined. Our findings suggest that PKC-β1 is important in the pathogenesis of pulmonary fibrosis.

T1α/Podoplanin Shows Raft-Associated Distribution in Mouse Lung Alveolar Epithelial E10 Cells

Aims: T1α/(podoplanin) is abundantly expressed in the alveolar epithelial type I cells (ATI) of rodent and human lungs. Caveolin-1 is a classical primary structural protein of plasmalemal invaginations, so-called caveolae, which represent specialized lipid rafts, and which are particularly abundant in ATI cells. The biological functions of T1α in the alveolar epithelium are unknown. Here we report on the characteristics of raft domains in the microplicae/microvillar protrusions of ATI cells, which contain T1α. Methods: Detergent resistant membranes (DRMs) from cell lysates of the mouse epithelial ATI-like cell line E10 were prepared using different detergents followed by flotation in a sucrose gradient and tested by Western and dot blots with raft markers (caveolin-1, GM1) and nonraft markers (transferrin receptor, PDI and β-Cop). Immunocytochemistry was employed for the localization of T1α in E10 cells and in situ in rat lungs. Results: Our biochemical results showed that the solubility or insolubility of T1α and caveolin-1 differs in Triton X-100 and Lubrol WX, two distinct non-ionic detergents. Caveolin-1 was unsoluble in both detergents, whereas T1α was Triton X-100 soluble but Lubrol WX insoluble. Immunofluorescence double stainings revealed that both proteins were colocalized with GM1, while caveolin-1 and T1α were not colocalized in the plasma membrane. Cholesterol depletion modified the segregation of T1α in Lubrol WX DRMs. Cellular processes in ultrathin sections of cultured mouse E10 cells were immunogold positive. Immunoelectron microscopy (postembedding) of rat lung tissue revealed the preferential localization of T1α on apical microvillar protrusions of ATI cells. Conclusion: We conclude that T1α and caveolin-1 are located in distinct plasma membrane microdomains, which differ in their protein-lipid interactions. The raft-associated distribution of T1α may have an impact on a specific, not yet clarified function of this protein in the alveolar epithelium.