The origin of brain malignancies at the blood-brain barrier.

Despite improvements in extracranial therapy, survival rate for patients suffering from brain metastases remains very poor. This is coupled with the incidence of brain metastases continuing to rise. In this review, we focus on core contributions of the blood-brain barrier to the origin of brain metastases. We first provide an overview of the structure and function of the blood-brain barrier under physiological conditions. Next, we discuss the emerging idea of a pre-metastatic niche, namely that secreted factors and extracellular vesicles from a primary tumor site are able to travel through the circulation and prime the neurovasculature for metastatic invasion. We then consider the neurotropic mechanisms that circulating tumor cells possess or develop that facilitate disruption of the blood-brain barrier and survival in the brain's parenchyma. Finally, we compare and contrast brain metastases at the blood-brain barrier to the primary brain tumor, glioma, examining the process of vessel co-option that favors the survival and outgrowth of brain malignancies.

EGFL7 loss correlates with increased VEGF-D expression, upregulating hippocampal adult neurogenesis and improving spatial learning and memory.

Neural stem cells reside in the subgranular zone, a specialized neurogenic niche of the hippocampus. Throughout adulthood, these cells give rise to neurons in the dentate gyrus, playing an important role in learning and memory. Given that these core cognitive processes are disrupted in numerous disease states, understanding the underlying mechanisms of neural stem cell proliferation in the subgranular zone is of direct practical interest. Here, we report that mature neurons, neural stem cells and neural precursor cells each secrete the neurovascular protein epidermal growth factor-like protein 7 (EGFL7) to shape this hippocampal niche. We further demonstrate that EGFL7 knock-out in a Nestin-CreERT2-based mouse model produces a pronounced upregulation of neurogenesis within the subgranular zone. RNA sequencing identified that the increased expression of the cytokine VEGF-D correlates significantly with the ablation of EGFL7. We substantiate this finding with intraventricular infusion of VEGF-D upregulating neurogenesis in vivo and further show that VEGF-D knock-out produces a downregulation of neurogenesis. Finally, behavioral studies in EGFL7 knock-out mice demonstrate greater maintenance of spatial memory and improved memory consolidation in the hippocampus by modulation of pattern separation. Taken together, our findings demonstrate that both EGFL7 and VEGF-D affect neurogenesis in the adult hippocampus, with the ablation of EGFL7 upregulating neurogenesis, increasing spatial learning and memory, and correlating with increased VEGF-D expression.

P2X7 Receptor Indirectly Regulates the JAM-A Protein Content via Modulation of GSK-3ß.

The alveolar epithelial cells represent an important part of the alveolar barrier, which is maintained by tight junction proteins, particularly JAM-A, occludin, and claudin-18, which regulate paracellular permeability. In this study, we report on a strong increase in epithelial JAM-A expression in P2X7 receptor knockout mice when compared to the wildtype. Precision-cut lung slices of wildtype and knockout lungs and immortal epithelial lung E10 cells were treated with bleomycin, the P2X7 receptor inhibitor oxATP, and the agonist BzATP, respectively, to evaluate early changes in JAM-A expression. Biochemical and immunohistochemical data showed evidence for P2X7 receptor-dependent JAM-A expression in vitro. Inhibition of the P2X7 receptor using oxATP increased JAM-A, whereas activation of the receptor decreased the JAM-A protein level. In order to examine the role of GSK-3ß in the expression of JAM-A in alveolar epithelial cells, we used lithium chloride for GSK-3ß inhibiting experiments, which showed a modulating effect on bleomycin-induced alterations in JAM-A levels. Our data suggest that an increased constitutive JAM-A protein level in P2X7 receptor knockout mice may have a protective effect against bleomycin-induced lung injury. Bleomycin-treated precision-cut lung slices from P2X7 receptor knockout mice responded with a lower increase in mRNA expression of JAM-A than bleomycin-treated precision-cut lung slices from wildtype mice.

Neurodegeneration and Neuro-Regeneration-Alzheimer's Disease and Stem Cell Therapy.

Aging causes many changes in the human body, and is a high risk for various diseases. Dementia, a common age-related disease, is a clinical disorder triggered by neurodegeneration. Brain damage caused by neuronal death leads to cognitive decline, memory loss, learning inabilities and mood changes. Numerous disease conditions may cause dementia; however, the most common one is Alzheimer's disease (AD), a futile and yet untreatable illness. Adult neurogenesis carries the potential of brain self-repair by an endogenous formation of newly-born neurons in the adult brain; however it also declines with age. Strategies to improve the symptoms of aging and age-related diseases have included different means to stimulate neurogenesis, both pharmacologically and naturally. Finally, the regulatory mechanisms of stem cells neurogenesis or a functional integration of newborn neurons have been explored to provide the basis for grafted stem cell therapy. This review aims to provide an overview of AD pathology of different neural and glial cell types and summarizes current strategies of experimental stem cell treatments and their putative future use in clinical settings.

P2X7R: independent modulation of aquaporin 5 expression in CdCl2-injured alveolar epithelial cells.

The expression of aquaporin 5 in alveolar epithelial type I cells under conditions of cadmium-induced injury has not yet been discovered. We investigated the effect of the P2X7R agonist BzATP under this condition, since P2X7R is involved in altered regulation of aquaporin 5 in pulmonary fibrosis. CdCl2/TGF-ß1 treatment of lung epithelial MLE-12 cells was leading to increasing P2X7R, and aquaporin 5 protein levels. The aquaporin 5 expression was P2X7R-independent in MLE-12 cells under cadmium, as was shown in blocking experiments with oxATP. Further, the expression of both proteins increased after 24 h CdCl2/TGF-ß1 treatment of precision-cut lung slices, but decreased after 72 h. Using immunohistochemistry, the activation of the P2X7R with the agonist BzATP modulated the aquaporin 5 immunoreactivity in the alveolar epithelium of precision-cut lung slices from wild-type but not from P2X7R knockout mice. Similarly, aquaporin 5 protein was reduced in BzATP-treated immortal lung epithelial E10 cells. Surprisingly, untreated alveolar epithelial type II cells of P2X7R knockouts exhibited a pronounced apical immunoreactivity in addition to the remaining alveolar epithelial type I cells. BzATP exposure did not alter this distribution pattern, but increased the number of apoptotic alveolar epithelial type II cells in wild-type lung slices.

Phospho-NHE3 forms membrane patches and interacts with beta-actin to sense and maintain constant direction during cell migration.

The Na(+)/H(+) exchanger NHE3 colocalizes with beta-actin at the leading edge of directionally migrating cells. Using human osteosarcoma cells (SaOS-2), rat osteoblasts (calvaria), and human embryonic kidney (HEK) cells, we identified a novel role for NHE3 via beta-actin in anode and cathode directed motility, during electrotaxis. NHE3 knockdown by RNAi revealed that NHE3 expression is required to achieve constant directionality and polarity in migrating cells. Phosphorylated NHE3 (pNHE3) and beta-actin complex formation was impaired by the NHE3 inhibitor S3226 (IC50 0.02µM). Fluorescence cross-correlation spectroscopy (FCCS) revealed that the molecular interactions between NHE3 and beta-actin in membrane protrusions increased 1.7-fold in the presence of a directional cue and decreased 3.3-fold in the presence of cytochalasin D. Data from flow cytometric analysis showed that membrane potential of cells (Vmem) decreases in directionally migrating, NHE3-deficient osteoblasts and osteosarcoma cells whereas only Vmem of wild type osteoblasts is affected during directional migration. These findings suggest that pNHE3 has a mechanical function via beta-actin that is dependent on its physiological activity and Vmem. Furthermore, phosphatidylinositol 3,4,5-trisphosphate (PIP3) levels increase while PIP2 remains stable when cells have persistent directionality. Both PI3 kinase (PI3K) and Akt expression levels change proportionally to NHE3 levels. Interestingly, however, the content of pNHE3 level does not change when PI3K/Akt is inhibited. Therefore, we conclude that NHE3 can act as a direction sensor for cells and that NHE3 phosphorylation in persistent directional cell migration does not involve PI3K/Akt during electrotaxis.

c-Cbl Regulates Murine Subventricular Zone-Derived Neural Progenitor Cells in Dependence of the Epidermal Growth Factor Receptor.

The localization, expression, and physiological role of regulatory proteins in the neurogenic niches of the brain is fundamental to our understanding of adult neurogenesis. This study explores the expression and role of the E3-ubiquitin ligase, c-Cbl, in neurogenesis within the subventricular zone (SVZ) of mice. In vitro neurosphere assays and in vivo analyses were performed in specific c-Cbl knock-out lines to unravel c-Cbl's role in receptor tyrosine kinase signaling, including the epidermal growth factor receptor (EGFR) pathway. Our findings suggest that c-Cbl is significantly expressed within EGFR-expressing cells, playing a pivotal role in neural stem cell proliferation and differentiation. However, c-Cbl's function extends beyond EGFR signaling, as its loss upon knock-out stimulated progenitor cell proliferation in neurosphere cultures. Yet, this effect was not detected in hippocampal progenitor cells, reflecting the lack of the EGFR in the hippocampus. In vivo, c-Cbl exerted only a minor proneurogenic influence with no measurable impact on the formation of adult-born neurons. In conclusion, c-Cbl regulates neural stem cells in the subventricular zone via the EGFR pathway but, likely, its loss is compensated by other signaling modules in vivo.

Detection of caveolin-3/caveolin-1/P2X7R complexes in mice atrial cardiomyocytes in vivo and in vitro.

Caveolae and caveolins, structural components of caveolae, are associated with specific ion channels in cardiac myocytes. We have previously shown that P2X purinoceptor 7 (P2X7R), a ligand-gated ion channel, is increased in atrial cardiomyocytes of caveolin-1 knockout mice; however, the specific biochemical relationship of P2X7R with caveolins in the heart is not clear. The aim of this work was to study the presence of the P2X7R in atrial cardiomyocytes and its biochemical relationship to caveolin-1 and caveolin-3. Caveolin isoforms and P2X7R were predominantly localized in buoyant membrane fractions (lipid rafts/caveolae) prepared from hearts using detergent-free sucrose gradient centrifugation. Caveolin-1 knockout mice showed normal distribution of caveolin-3 and P2X7R to buoyant membranes indicating the importance of caveolin-3 to formation of caveolae. Using clear native-PAGE, we showed that caveolin-1, -3 and P2X7R contribute to the same protein complex in the membranes of murine cardiomyocytes and in the immortal cardiomyocyte cell line HL-1. Western blot analysis revealed increased caveolin-1 and -3 proteins in tissue homogenates of P2X7R knockout mice. Finally, tissue homogenates of atrial tissues from caveolin-3 knockout mice showed elevated mRNA for P2X7R in atria. The colocalization of caveolins with P2X7R in a biochemical complex and compensated upregulation of P2X7R or caveolins in the absence of any component of the complex suggests P2X7R and caveolins may serve an important regulatory control point for disease pathology in the heart.

Fyn-kinase and caveolin-1 in the alveolar epithelial junctional adherence complex contribute to the early stages of pulmonary fibrosis.

Current pathophysiological findings indicate that damage to the alveolar epithelium plays a decisive role in the development of idiopathic pulmonary fibrosis (IPF). The available pharmacological interventions (i.e., oral pirfenidone and nintedanib) only slow down progression of the disease, but do not offer a cure. In order to develop new drug candidates, the pathophysiology of IPF needs to be better understood on a molecular level. It has previously been reported that a loss of caveolin-1 (Cav-1) contributes to profibrotic processes by causing reduced alveolar barrier function and fibrosis-like alterations of the lung-parenchyma. Conversely, overexpression of caveolin-1 appears to counteract the development of fibrosis by inhibiting the inflammasome NLRP3 and the associated expression of interleukin-1ß. In this study, the interaction between Fyn-kinase and caveolin-1 in the alveolar epithelium of various bleomycin (BLM)/TGF-ß damage models using precision-cut lung slices (PCLS), wildtype (WT) and caveolin-1 knockout (KO) mice as well as the human NCI-H441 cell line, were investigated. In WT mouse lung tissues, strong signals for Fyn-kinase were detected in alveolar epithelial type I cells, whereas in caveolin-1 KO animals, expression shifted to alveolar epithelial type II cells. Caveolin-1 and Fyn-kinase were found to be co-localized in isolated lipid rafts of NCI-H441 cell membrane fractions. These findings were corroborated by co-immunoprecipitation studies in which a co-localization of Cav-1 and Fyn-kinase was detected in the cell membrane of the alveolar epithelium. After TGF-ß and BLM-induced damage to the alveolar epithelium both in PCLS and cell culture experiments, a decrease in caveolin-1 and Fyn-kinase was found. Furthermore, TEER (transepithelial electrical resistance) measurements indicated that TGF-ß and BLM have a damaging effect on cell-cell contacts and thus impair the barrier function in NCI-H441 cell monolayers. This effect was attenuated after co-incubation with the Fyn-kinase inhibitor, PP-2. Our data suggest an involvement of Fyn-kinase and caveolin-1 in TGF-ß/bleomycin-induced impairment of alveolar barrier function and thus a possible role in the early stages of pulmonary fibrosis. Fyn-kinase and/or its complex with caveolin-1 might, therefore, be novel therapeutic targets in IPF.

Interaction and interrelation of P2X7 and P2X4 receptor complexes in mouse lung epithelial cells.

P2X4 and P2X7 receptors are ATP-gated ion channels that are co-expressed in alveolar epithelial type I cells. Both receptors are localized to the plasma membrane and partly associated with lipid rafts. Here we report on our study in an alveolar epithelial cell line of the molecular organization of P2X7R and P2X4R receptors and the effect of their knockdown. Native gel electrophoresis reveals three P2X7R complexes of approximately 430, approximately 580 and approximately 760 kDa. The latter two correspond exactly in size to signals of Cav-1, the structural protein of caveolae. Interestingly knockdown of P2rx7 affects protein levels, the intracellular distribution and the supramolecular organization of Cav-1 as well as of P2X4R, which is mainly detected in a complex of approximately 430 kDa. Our data suggest upregulation of P2X4R as a compensatory mechanism of P2X7R depletion.

T1alpha/podoplanin shows raft-associated distribution in mouse lung alveolar epithelial E10 cells.

AIMS: T1alpha/(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 T1alpha 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 T1alpha. 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 beta-Cop). Immunocytochemistry was employed for the localization of T1alpha in E10 cells and in situ in rat lungs. RESULTS: Our biochemical results showed that the solubility or insolubility of T1alpha and caveolin-1 differs in Triton X-100 and Lubrol WX, two distinct non-ionic detergents. Caveolin-1 was unsoluble in both detergents, whereas T1alpha was Triton X-100 soluble but Lubrol WX insoluble. Immunofluorescence double stainings revealed that both proteins were colocalized with GM1, while caveolin-1 and T1alpha were not colocalized in the plasma membrane. Cholesterol depletion modified the segregation of T1alpha 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 T1alpha on apical microvillar protrusions of ATI cells. CONCLUSION: We conclude that T1alpha and caveolin-1 are located in distinct plasma membrane microdomains, which differ in their protein-lipid interactions. The raft-associated distribution of T1alpha may have an impact on a specific, not yet clarified function of this protein in the alveolar epithelium.

Biopsies of olfactory epithelium in patients with Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder involving several neuronal systems. Impaired olfactory function may constitute one of the earliest symptoms of PD. However, it is still unclear to what degree changes of the olfactory epithelium may contribute to dysosmia and if these changes are different from those of other hyposmic or anosmic patients. This study aimed to investigate the hypothesis that olfactory loss in PD is a consequence of specific PD-related damage of olfactory epithelium. Biopsies of 7 patients diagnosed with PD were taken. Six patients with PD were hyposmic, one anosmic. As non-PD controls served 9 patients with hyposmia, 9 with anosmia, and 7 normosmic individuals. Further, nasal mucosa of 4 postmortem individuals was investigated. Immunohistochemical examinations were performed with antibodies against olfactory marker protein (OMP), protein gene product 9.5 (PGP 9.5), beta-tubulin, (BT), proliferation-associated antigen (Ki 67), the stem cell marker nestin, cytokeratin, p75NGFr, and alpha-synuclein. Most of the biopsy specimens exhibited irregular areas of olfactory-like, dysplastic epithelium positive for either PGP 9.5 or BT, but negative for OMP. No major histochemical differences in either the expression or distribution of these proteins were observed in the olfactory epithelium of patients with PD compared with controls. Reverse transcription PCR (RT-PCR) data indicated mRNA for OMP in almost all subjects, independently of their olfactory performance. These data support the idea that olfactory loss in Parkinson's disease is not a consequence of damage to the olfactory epithelium but rather results from distinct central-nervous abnormalities.

Downregulation of caveolin-1 affects bleomycin-induced growth arrest and cellular senescence in A549 cells.

Bleomycin is an anti-cancer drug that induces both apoptosis and senescence, two processes thought to involve caveolin-1. Here we investigate the role of caveolin-1 in bleomycin-induced senescence. We show that bleomycin-treated A549 cells exhibit: senescence-like cell morphology; a senescence-associated increase in SA-beta-galactosidase activity; cell cycle arrest; and upregulation of p53 and p21. As predicted, we find that caveolin-1 amount increases in response to bleomycin-treatment and that modulation of caveolin-1 affects p21 and p53 levels, cell cycling, and senescence (SA-beta-galactosidase activity). Interestingly, senescence-associated cell cycle arrest via p53 and p21 and SA-beta-galactosidase activity is reduced in young A549 cells when short hairpin RNA specific for caveolin-1 was applied before bleomycin-treatment. Our results support the hypothesis that downregulation of caveolin-1 expression affects bleomycin-induced cell cycle arrest and subsequent cellular senescence that is driven by p53 and p21.

Bleomycin induces caveolin-1 and -2 expression in epithelial lung cancer A549 cells.

BACKGROUND: Bleomycin induces apoptosis in alveolar epithelial cells. The expression of caveolin-1 and -2 in lung epithelial-derived A549 cells was analysed in terms of apoptosis after exposure to bleomycin. MATERIALS AND METHODS: Apoptosis was investigated using flow cytometry, ELISA, immunohistochemistry and Western blot analysis. Caveolin-1 and -2 were determined at the protein level (Western blot). Intracellular caveolin-1 distribution was studied with immunofluorescence, as well as sucrose density gradient centrifugation. RESULTS: Caveolin-1 and -2 were up-regulated 1 h after exposure to bleomycin and preceding the occurrence of caspase-8, and of caspase-3 and caspase-9 cleavage products. Sucrose density gradient centrifugation revealed that bleomycin exposure led to a partial translocation of caveolin-1 from caveolin-rich membrane fractions to non-raft fractions. Successful inhibition of bleomycin-induced apoptosis by the broad-spectrum caspase inhibitor zVAD-fmk did not influence the amount of caveolin-1 and -2. CONCLUSION: The early up-regulation of caveolin-1 and -2 following bleomycin exposure is a rather apoptosis-independent event related to other unknown mechanisms of bleomycin-mediated cell injury.

Potential contribution of alveolar epithelial type I cells to pulmonary fibrosis.

Pulmonary fibrosis (PF) is characterized by inflammation and fibrosis of the interstitium and destruction of alveolar histoarchitecture ultimately leading to a fatal impairment of lung function. Different concepts describe either a dominant role of inflammatory pathways or a disturbed remodeling of resident cells of the lung parenchyma during fibrogenesis. Further, a combination of both the mechanisms has been postulated. The present review emphasizes the particular involvement of alveolar epithelial type I cells in all these processes, their contribution to innate immune/inflammatory functions and maintenance of proper alveolar barrier functions. Amongst the different inflammatory and repair events the purinergic receptor P2X7, an ATP-gated cationic channel that regulates not only apoptosis, necrosis, autophagy, and NLPR3 inflammosome activation, but also the turnover of diverse tight junction (TJ) and water channel proteins, seems to be essential for the stability of alveolar barrier integrity and for the interaction with protective factors during lung injury.

A novel role of endothelium in activation of latent pro-membrane type 1 MMP and pro-MMP-2 in rat aorta.

AIMS: Aortic stiffness is an independent risk factor for progression of cardiovascular diseases. Degradation of elastic fibres in aorta due to angiotensin II (ANGII)-stimulated overactivation of latent membrane type 1 matrix metalloproteinase (MT1MMP) and matrix metalloproteinase-2 (MMP2) is regarded to represent an important cause of aortic stiffness. Therefore, clarification of the causal mechanisms triggering the overactivation of these MMPs is of utmost importance. This study addresses the endothelium as a novel key activator of latent pro-MT1MMP and pro-MMP2 in rat aorta. METHODS AND RESULTS: Using a co-culture model of rat aortic endothelial cells (ECs) and smooth muscle cells (SMCs), we found that ANGII stimulation resulted in activation of latent pro-MT1MMP and pro-MMP2 in SMCs exclusively when co-cultured with ECs (assessed with western blot and gelatin zymography, respectively). EC-specific AT1 receptor stimulation triggered endothelin-1 release and paracrine action on SMCs. Endothelin-1 increased expression and activity of pro-protein convertase furin in SMCs via endothelin receptor type A (assessed with qPCR and furin activity assay, respectively). Consequently, furin acted in two ways. First, it increased the activation of latent pro-MT1MMP and, second, it activated pro-αvß3 integrin. Both pathways led to overactivation of latent pro-MMP2. In vitro findings in the co-culture model were fully consistent with the ex vivo findings obtained in isolated rat aorta. CONCLUSIONS: We propose that the endothelium under ANGII stimulation acts as a novel and key activator of latent pro-MT1MMP and pro-MMP2 in SMCs of rat aorta. Therefore, endothelium may critically contribute to pathophysiology of aortic stiffness.

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-ß1, 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.

Effect of P2X7 receptor knockout on AQP-5 expression of type I alveolar epithelial cells.

P2X7 receptors, ATP-gated cation channels, are specifically expressed in alveolar epithelial cells. The pathophysiological function of this lung cell type, except a recently reported putative involvement in surfactant secretion, is unknown. In addition, P2X7 receptor-deficient mice show reduced inflammation and lung fibrosis after exposure with bleomycin. To elucidate the role of the P2X7 receptor in alveolar epithelial type I cells we characterized the pulmonary phenotype of P2X7 receptor knockout mice by using immunohistochemistry, western blot analysis and real-time RT PCR. No pathomorphological signs of fibrosis were found. Results revealed, however, a remarkable loss of aquaporin-5 protein and mRNA in young knockout animals. Additional in vitro experiments with bleomycin treated precision cut lung slices showed a greater sensitivity of the P2X7 receptor knockout mice in terms of aquaporin-5 reduction as wild type animals. Finally, P2X7 receptor function was examined by using the alveolar epithelial cell lines E10 and MLE-12 for stimulation experiments with bleomycin. The in vitro activation of P2X7 receptor was connected with an increase of aquaporin-5, whereas the inhibition of the receptor with oxidized ATP resulted in down regulation of aquaporin-5. The early loss of aquaporin-5 which can be found in different pulmonary fibrosis models does not implicate a specific pathogenetic role during fibrogenesis.

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-ß1 levels. BLM treatment or stimulation of the P2X7R with the P2X7R agonist BzATP induced translocation of PKC-ß1 from the cytoplasm to the membrane. The expression of PKC-ß1 was repressed by the P2X7R inhibitor oxATP, suggesting that PKC-ß1 is downstream of P2X7R activation. Furthermore, cells exposed to BLM contained increased amounts of P2X7R and PKC-ß1 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-ß1 and CaM as well as decreased immunoreactivity for PKC-ß1. The knockdown of P2X7R in alveolar epithelial cells resulted also in a loss of PKC-ß1. These data suggest that the effect of P2X7R on expression of PKC-ß1 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-ß1 immunoreactivity. The present experiments demonstrated that the increased expression of P2X7R influences PKC-ß1. We predict that increased Ca(2+) concentration stimulates PKC-ß1, whereas the prerequisite for activating PKC-ß1 after P2X7R increase remained to be determined. Our findings suggest that PKC-ß1 is important in the pathogenesis of pulmonary fibrosis.