ENaC Dysregulation Through Activation of MEK1/2 Contributes to Impaired Na+ Absorption in Lymphocytic Colitis.

BACKGROUND: Lymphocytic colitis (LC) causes watery diarrhea. We aimed to identify mechanisms of altered Na absorption and regulatory inputs in patients with LC by examining the epithelial Na channel (ENaC) function as the predominant Na transport system in human distal colon. METHODS: Epithelial Na channel function and regulation was analyzed in biopsies from sigmoid colon of patients with LC and in rat distal colon in Ussing chambers. ENaC-subunit expression was measured by real-time PCR and RNA sequencing. Correction factors for subepithelial resistance contributions were determined by impedance spectroscopy. Upstream regulators in LC were determined by RNA sequencing. RESULTS: Epithelial Na channel-mediated electrogenic Na transport was inhibited despite aldosterone stimulation in human sigmoid colon of patients with LC. The increase in γ-ENaC mRNA expression in response to aldosterone was MEK1/2-dependently reduced in LC, since it could be restored toward normal by MEK1/2 inhibition through U0126. Parallel experiments for identification of signaling in rat distal colon established MEK1/2 to be activated by a cytokine cocktail of TNFα, IFNγ, and IL-15, which were identified as the most important regulators in the upstream regulator analysis in LC. CONCLUSIONS: In the sigmoid colon of patients with LC, the key effector cytokines TNFα, IFNγ, and IL-15 inhibited γ-ENaC upregulation in response to aldosterone through a MEK1/2-mediated pathway. This prevents ENaC to reach its maximum transport capacity and results in Na malabsorption which contributes to diarrhea.

Interleukin-13 affects the epithelial sodium channel in the intestine by coordinated modulation of STAT6 and p38 MAPK activity.

KEY POINTS: Interleukin-13 (IL-13) causes intestinal epithelial barrier dysfunction, and is implicated in the pathogenesis of Th2-driven intestinal inflammation (e.g. ulcerative colitis). However, it is unclear whether the epithelial sodium channel (ENaC) - the main limiting factor for sodium absorption in the distal colon - is also influenced by IL-13 and if so, by what mechanism(s). We demonstrate in an intestinal cell model as well as in mouse distal colon that IL-13 causes reduced ENaC activity. We show that IL-13 impairs ENaC-dependent sodium transport by activating the JAK1/2-STAT6 signalling pathway. These results improve our understanding of the mechanisms through which IL-13 functions as a key effector cytokine in ulcerative colitis, thereby contributing to the distinct pathology of this disease. ABSTRACT: Interleukin-13 (IL-13) has been strongly implicated in the pathogenesis of ulcerative colitis, possibly by disrupting epithelial integrity. In the distal colon, the epithelial sodium channel (ENaC) is an important factor in the regulation of sodium absorption, and therefore plays a critical role in minimizing intestinal sodium and water losses. In the present study, we investigated whether IL-13 also acts as a potent modulator of epithelial sodium transport via ENaC, and the signalling components involved. The effect of IL-13 on ENaC was examined in HT-29/B6-GR/MR human colon cells, as well as in mouse distal colon, by measuring amiloride-sensitive short-circuit current (ISC ) in Ussing chambers. The expression levels of ENaC subunits and the cellular components that contribute to ENaC activity were analysed by qRT-PCR and promoter gene assay. We show that IL-13, in both the cell model and in native intestinal tissue, impaired epithelial sodium absorption via ENaC (JNa ) as a result of decreased transcription levels of ß- and γ-ENaC subunits and SGK1, a post-translational regulator of ENaC activity, due to impaired promoter activity. The reduction in JNa was prevented by inhibition of JAK1/2-STAT6 signalling. This inhibition also affected the IL-13-induced decrease in p38 MAPK phosphorylation. The contribution of STAT6 to IL-13-mediated ENaC inactivation was confirmed in a STAT6(-/-) mouse model. In conclusion, these results indicate that IL-13, the levels of which are elevated in ulcerative colitis, contributes to impaired ENaC activity via modulation of the STAT6/p38 MAPK pathways.

The plant-derived glucocorticoid receptor agonist Endiandrin A acts as co-stimulator of colonic epithelial sodium channels (ENaC) via SGK-1 and MAPKs.

In a search for secondary plant compounds that bind to the glucocorticoid receptor (GR), the cyclobutane lignan endiandrin A was discovered from the rainforest tree Endiandra anthropophagorum Domin. Our present study aims to characterize the effect of endiandrin A on GR-dependent induction of colonic sodium transport. The effect of endiandrin A was analyzed in GR-expressing colonic HT-29/B6 cells (HT-29/B6-GR). GR transactivation and subcellular localization were investigated by reporter gene assay and immunofluorescence. Epithelial sodium channel (ENaC) was analyzed by qRT-PCR and by measuring amiloride-sensitive short-circuit current (I(sc)) in Ussing chambers. Endiandrin A (End A) has been identified as GR receptor binder. However, it did not cause significant GR transactivation as pGRE-luciferase activity was only 7% of that of the maximum effect of dexamethasone. Interestingly, endiandrin A had a significant impact on dexamethasone-dependent sodium absorption in cells co-exposed to tumor necrosis factor (TNF)-α. This was in part due to up-regulation of ß- and γ-ENaC subunit expression. Endiandrin A potentiated GR-mediated transcription by increasing GR protein expression and phosphorylation. It inhibited c-Jun N-terminal kinase (JNK) activation induced by dexamethasone and/or TNF-α and increased levels of GR localized to the nucleus. Additionally, endiandrin A increased the serum- and glucocorticoid-induced kinase (sgk)-1 via activation of p38. Finally, the regulation of ENaC function by endiandrin A was confirmed in rat native colon. In conclusion, endiandrin A potentiates glucocorticoid-driven activation of colonic epithelial sodium channels via JNK inhibition and p38 activation due to transcriptional up-regulation of ß- and γ-ENaC-subunits along with induction of sgk-1.

No non-redundant function of suppressor of cytokine signaling 2 in insulin producing ß-cells.

The members of the Suppressor of Cytokine Signaling (SOCS) protein family mainly modulate the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. SOCS-1 and SOCS-3 have already been shown to influence growth and apoptosis of pancreatic beta cells. We hypothesized that SOCS-2, which is expressed in pancreatic islets, also contributes to ß-cell physiology. We tested this hypothesis in vivo in SOCS-2-/- knockout mice and in vitro in Ins-1E rat insulinoma cells. We found that SOCS-2-/- mice have normal islet insulin secretion and unchanged glucose and insulin tolerance compared to wildtype controls. SOCS-2-/- are bigger than wildtype mice but body weight-corrected ß-cell mass and islet morphology were normal. Growth hormone-induced proliferation of Ins-1E cells was not affected by either siRNA-mediated SOCS-2 knockdown or stable SOCS-2 overexpression. Interleukin-1ß mediated cell death in vitro was unchanged after SOCS-2 knockdown. Similarly, autoimmune destruction of beta cells in vivo after multiple low-dose injections of streptozotocin (STZ) was not altered in SOCS-2-/- mice. In summary, SOCS-2-/- knockout mice have a normal function of insulin-producing pancreatic ß-cells, a fully adapted beta cell mass and a normal morphology of the endocrine islets. Based on in vitro evidence, the increased ß-cell mass in the mutants is likely due to indirect adaptive mechanisms and not the result of altered growth hormone signaling within the ß-cells. Immune mediated ß-cell destruction is also not affected by SOCS-2 ablation in vitro and in vivo.

Relative roles of the different Pax6 domains for pancreatic alpha cell development.

BACKGROUND: The transcription factor Pax6 functions in the specification and maintenance of the differentiated cell lineages in the endocrine pancreas. It has two DNA binding domains, the paired domain and the homeodomain, in addition to a C-terminal transactivation domain. The phenotype of Pax6-/- knockout mice suggests non-redundant functions of the transcription factor in the development of glucagon-expressing alpha-cells as this cell type is absent in the mutants. We ask the question of how the differentiation of pancreatic endocrine cells, in particular that of alpha-cells, is affected by selective inactivation of either one of the three major domains of Pax6. RESULTS: The Pax6Aey18 mutant mouse line, in which the paired domain is inactivated, showed a phenotype similar to that of Pax6-/- knockout mice with a near complete absence of glucagon-positive alpha-cells (0-4 cells/section; < or =1% of wt), reduced beta-cell area (74% of wt) and disorganized islets. The proportion of ghrelin-positive epsilon-cells was expanded. In Pax6Sey-Neu mutants, which lack the transactivation domain, alpha-and beta-cells where reduced to 25 and 40% of wt, respectively. We also studied two mouse lines with mutations in the homeodomain, Pax64Neu and Pax6132-14Neu. Neighboring amino acids are affected in the two lines and both point mutations abolish DNA binding of the classical P3 homeodomain target sequence. The pancreatic phenotype of the two mutants however was divergent. While Pax64Neu homozygotes showed a reduction of alpha- and beta-cells to 59 and 61%, respectively, pancreatic endocrine development was unaltered in the Pax6132-14Neu mutant strain. CONCLUSIONS: We show that inactivation of the Pax6 paired domain leads to a more severe phenotype with regards to the differentiation of pancreatic alpha-cells than the loss of the transactivation domain. The analysis of two different homeodomain mutants suggests that the binding of Pax6 to P3 homeodomain consensus sequences is not required for alpha-cell development. It rather seems that the homeodomain has a modulating role in Pax6 function, possibly by facilitating a PH0-like binding confirmation on paired domain target genes like proglucagon. This function is differentially affected by the two homeodomain mutations analyzed in this study.

The effect of CpG motifs on gene expression and clearance kinetics of aerosol administered polyethylenimine (PEI)-plasmid DNA complexes in the lung.

Presence of CpG motifs within pDNA is widely reported to influence transgene expression as well as inflammatory response to nonviral gene vector complexes. Here, we analyzed gene expression kinetics and lung clearance after aerosol delivery of polyethylenimine (PEI) complexes with two different plasmid vectors: a first generation plasmid, pCMVLuc, and a plasmid with depleted CpG motifs, pCpG-free-Luc. After aerosol delivery, equal nanogram amounts of PEI-pDNA complexes were deposited in murine lungs. Luciferase expression observed at day one post administration of PEI-pCpG-free-Luc complexes was 60-fold higher than for PEI-pCMVLuc complexes and decreased 16-fold at day 7 post application. In contrast, luciferase expression from PEI-pCMVLuc particles remained at levels comparable to day 1 post application. In agreement with these observations, PEI-pCpG-free-Luc complexes were cleared from the lungs at rates 6-fold faster than those observed for PEI-pCMVLuc particles. A more detailed analysis of pDNA distribution within bronchoalveolar lavage fluid (BALF), BALF cells and lung tissue showed 660-fold higher amounts of pCpG-free-Luc in BALF cells compared to pCMVLuc, whereas the amount of pCpG-free-Luc in lung tissue was 15-fold lower compared to pCMVLuc 1h after administration. Our results demonstrate that complexes of PEI with CpG-motif-free DNA are taken up more extensively by BALF cells, while the clearance of pCMVLuc from the lung tissue is significantly slower than for the CpG-free plasmid. Administration of PEI-pCpG-free-Luc caused transient decrease in number of resident lung cells, while their activation was more pronounced with PEI-pCMVLuc particles. Our results demonstrate that the level of transgene expression is increased with CpG-motif-free pDNA but the longevity of expression correlates with pDNA clearance pattern depending on the presence of CpG motifs within the plasmid.

CpG-free plasmid DNA prevents deterioration of pulmonary function in mice.

Nonviral gene vectors have been shown to be therapeutically effective in various animal models of inherited and acquired lung diseases. Although an acute unmethylated CG dinucleotide (CpG)-mediated inflammatory response has been previously observed for first-generation plasmids, its effect on pulmonary function has not been investigated to date. Here, we present data on lung functional parameters together with histopathology, cellular and inflammatory events in response to pulmonary administration of DNA-containing particles. We show that aerosol delivery of polyethylenimine gene vectors containing a first-generation CpG-rich plasmid induced an inflammatory response which was associated with a decrease in lung compliance. In contrast to these observations, aerosol application of CpG-free plasmid DNA prevented immune response and impairment of pulmonary function. These results demonstrate that aerosol delivery of CpG-free plasmid DNA is critical to avoid alteration of pulmonary function. Therefore, we suggest to use CpG-free pDNA for gene delivery to the lungs in future.

Targeting of the glucocorticoid hormone receptor with plasmid DNA comprising glucocorticoid response elements improves nonviral gene transfer efficiency in the lungs of mice.

BACKGROUND: It has been previously demonstrated that plasmid DNA transport into the nucleus could be increased by transcription factor binding. We chose the glucocorticoid responsive element (GRE) which binds to the glucocorticoid receptor (GR), a transcription factor which is shuttled into the nucleus upon ligand binding such as dexamethasone. METHODS: We cloned two, four, and eight repetitive sequences of the GRE into the reporter plasmid pEGFPLuc. Binding of the pEGFPLuc-GRE to the GR was examined by electrophoretic mobility shift assay (EMSA) experiments. GR expression in bronchiolar and alveolar epithelial cells was confirmed by Western blotting. Intracellular trafficking of GR was examined using a fusion protein of cyano-fluorescent protein (CFP) and GR. Transfection efficiencies of pEGFPLuc compared to pEGFPLucGRE(2-8) were examined in vitro and in vivo upon tail vein injection of cationic liposome gene vectors containing dexamethasone (safeplexes) and aerosol application of polyethylenimine (PEI)-pDNA particles. RESULTS: Binding of GRE containing plasmids to the GR was shown in EMSA experiments and intranuclear shuttling of CFP-GR after ligand stimulation was confirmed. Enhanced gene transfer efficiency of pEGFPLucGRE(2) in vitro was only observed on confluent cells. A 2.5-fold increase in gene expression in the lungs of mice after tail vein injection of pEGFPLucGRE(2) complexed with safeplexes compared with pEGFPLuc was observed. PEI-mediated aerosol gene delivery of pEGFPLucGRE(2) was 4.7-fold higher than pEGFPLuc only after intraperitoneal dexamethasone. CONCLUSION: The results suggest that inclusion of GRE sequences into plasmid DNA vectors combined with dexamethasone application could improve transgene expression in the lungs in vivo.

Aerosol gene delivery to the murine lung is mouse strain dependent.

The cationic polymer polyethylenimine (PEI) has been previously demonstrated to efficiently deliver genes to the lungs of mice in vivo via nebulization. Although within these studies various mouse strains were used in individual experiments, no direct comparison of gene delivery to different mouse strains via aerosol application has been published to date. With respect to the widespread use of mice as animal models of inherited and acquired diseases, such data could be of relevance to select the most appropriate mouse genetic background for preclinical mouse models. We investigated PEI-based aerosol gene delivery in two commonly used mouse strains, BALB/c and NMRI, and mixed 129/Sv x C57BL/6 mice. Gene expression in BALB/c mice was significantly 3.2- and 3.8-fold higher than in NMRI and 129/Sv x C57BL/6 mice, respectively. Lung deposition rates of radioactively labeled plasmid DNA (I(123)) complexed with PEI were not significantly different between each of the mouse strains. The kinetics of pDNA clearance from the lungs of BALB/c mice was slightly faster than from NMRI mice. Whereas gene expression increased until day 3 after treatment, the levels of pDNA decreased over the same period of time. Repeated aerosol application in a 3-day time interval could maintain gene expression at high levels compared with a single application. Furthermore, PEI-pDNA aerosol application led to reproducible gene expression in independent experiments. These data suggest that the genetic background of mice could be important for nonviral aerosol gene delivery which should be considered in transgenic animal mouse models of inherited and acquired diseases for aerosol gene delivery studies.

Targeted delivery of magnetic aerosol droplets to the lung.

The inhalation of medical aerosols is widely used for the treatment of lung disorders such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, respiratory infection and, more recently, lung cancer. Targeted aerosol delivery to the affected lung tissue may improve therapeutic efficiency and minimize unwanted side effects. Despite enormous progress in optimizing aerosol delivery to the lung, targeted aerosol delivery to specific lung regions other than the airways or the lung periphery has not been adequately achieved to date. Here, we show theoretically by computer-aided simulation, and for the first time experimentally in mice, that targeted aerosol delivery to the lung can be achieved with aerosol droplets comprising superparamagnetic iron oxide nanoparticles--so-called nanomagnetosols--in combination with a target-directed magnetic gradient field. We suggest that nanomagnetosols may be useful for treating localized lung disease, by targeting foci of bacterial infection or tumour nodules.

cAMP regulates plasma membrane vacuolar-type H+-ATPase assembly and activity in blowfly salivary glands.

Reversible assembly of the V0V1 holoenzyme from V0 and V1 subcomplexes is a widely used mechanism for regulation of vacuolar-type H+-ATPases (V-ATPases) in animal cells. In the blowfly (Calliphora vicina) salivary gland, V-ATPase is located in the apical membrane of the secretory cells and energizes the secretion of a KCl-rich saliva in response to the hormone serotonin. We have examined whether the cAMP pathway, known to be activated by serotonin, controls V-ATPase assembly and activity. Fluorescence measurements of pH changes at the luminal surface of isolated glands demonstrate that cAMP, Sp-adenosine-3',5'-cyclic monophosphorothioate, or forskolin, similar to serotonin, cause V-ATPase-dependent luminal acidification. In addition, V-ATPase-dependent ATP hydrolysis increases upon treatment with these agents. Immunofluorescence microscopy and pelleting assays have demonstrated further that V1 components become translocated from the cytoplasm to the apical membrane and V-ATPase holoenzymes are assembled at the apical membrane during conditions that increase intracellular cAMP. Because these actions occur without a change in cytosolic Ca2+, our findings suggest that the cAMP pathway mediates the reversible assembly and activation of V-ATPase molecules at the apical membrane upon hormonal stimulus.

Dopaminergic and serotonergic innervation of cockroach salivary glands: distribution and morphology of synapses and release sites.

The paired salivary glands in the cockroach are composed of acini with ion-transporting peripheral P-cells and protein-secreting central C-cells, and a duct system for the modification of the primary saliva. Secretory activity is controlled by serotonergic and dopaminergic neurons, whose axons form a dense plexus on the glands. The spatial relationship of release sites for serotonin and dopamine to the various cell types was determined by anti-synapsin immunofluorescence confocal microscopy and electron microscopy. Every C-cell apparently has only serotonergic synapses on its surface. Serotonergic and dopaminergic fibres on the acini have their release zones at a distance of approximately 0.5 microm from the P-cells. Nerves between acinar lobules may serve as neurohaemal organs and contain abundant dopaminergic and few serotonergic release sites. Some dopaminergic and serotonergic release sites reside in the duct epithelium, the former throughout the duct system, the latter only in segments next to acini. These findings are consistent with the view that C-cells respond exclusively to serotonin, P-cells to serotonin and dopamine, and most duct cells only to dopamine. Moreover, the data suggest that C-cells are stimulated by serotonin released close to their surface, whereas P-cells and most duct cells are exposed to serotonin/dopamine liberated at some distance.

Distribution and serotonin-induced activation of vacuolar-type H+-ATPase in the salivary glands of the blowfly Calliphora vicina.

Secretory activity in blowfly salivary glands is activated by the hormone serotonin. We have investigated the distribution and activity of two cation pumps that are possibly involved with transepithelial ion transport, i.e. Na(+)/K(+)-ATPase and vacuolar-type H(+)-ATPase (V-ATPase). By immunofluorescence labelling of secretory cells, Na(+)/K(+)-ATPase was localized on the basolateral plasma membrane and V-ATPase on the highly folded apical membrane. Activities of both ATPases were probed in salivary gland homogenates by applying specific inhibitors for these ion pumps, namely ouabain and bafilomycin A(1). In control glands, bafilomycin-A(1)-sensitive V-ATPase activity and ouabain-sensitive Na(+)/K(+)-ATPase activity accounted for 36% and 19%, respectively, of the total ATPase activity. V-ATPase activity increased approximately twofold after stimulation with serotonin, whereas Na(+)/K(+)-ATPase activity was not significantly affected. Biochemical assays provided evidence that the serotonin-induced activation of V-ATPase activity was accompanied by a recruitment of peripheral V(1) subunits from the cytosol to the plasma membrane, indicative of the assembly of V(0)V(1) holoenzymes. These data show that a V-ATPase located in the apical plasma membranes of the secretory cells is a component of the apical "potassium pump" that has been identified previously by physiological approaches. The V-ATPase energizes the apical membrane and provides the primary driving force for fuelling a putative K(+)/nH(+) antiporter and, thus, for fluid secretion. Serotonin-induced assembly of V(0)V(1) holoenzymes might constitute a regulatory mechanism for the control of pump activity.

Distribution of serotonergic and dopaminergic nerve fibers in the salivary gland complex of the cockroach Periplaneta americana.

BACKGROUND: The cockroach salivary gland consists of secretory acini with peripheral ion-transporting cells and central protein-producing cells, an extensive duct system, and a pair of reservoirs. Salivation is controlled by serotonergic and dopaminergic innervation. Serotonin stimulates the secretion of a protein-rich saliva, dopamine causes the production of a saliva without proteins. These findings suggest a model in which serotonin acts on the central cells and possibly other cell types, and dopamine acts selectively on the ion-transporting cells. To examine this model, we have analyzed the spatial relationship of dopaminergic and serotonergic nerve fibers to the various cell types. RESULTS: The acinar tissue is entangled in a meshwork of serotonergic and dopaminergic varicose fibers. Dopaminergic fibers reside only at the surface of the acini next to the peripheral cells. Serotonergic fibers invade the acini and form a dense network between central cells. Salivary duct segments close to the acini are locally associated with dopaminergic and serotonergic fibers, whereas duct segments further downstream have only dopaminergic fibers on their surface and within the epithelium. In addition, the reservoirs have both a dopaminergic and a serotonergic innervation. CONCLUSION: Our results suggest that dopamine is released on the acinar surface, close to peripheral cells, and along the entire duct system. Serotonin is probably released close to peripheral and central cells, and at initial segments of the duct system. Moreover, the presence of serotonergic and dopaminergic fiber terminals on the reservoir indicates that the functions of this structure are also regulated by dopamine and serotonin.