Jane Sun

Purinergic inhibition of the epithelial Na+ transport via hydrolysis of PIP2

Stimulation of purinergic receptors inhibits amiloride‐sensitive Na+ transport in epithelial tissues by an unknown mechanism. Because previous studies excluded the role of intracellular Ca2+ or protein kinase C, we examined whether purinergic regulation of Na+ absorption occurs via hydrolysis of phospholipid such as phosphatidylinositol‐bisphosphates (PIP2). Inhibition of amiloride‐sensitive short‐circuit currents (Isc‐Amil) by adenine 5′‐triphosphate (ATP) in native tracheal epithelia and M1 collecting duct cells was suppressed by binding neomycin to PIP2, and recovery from ATP inhibition was abolished by blocking phosphatidylinositol‐4‐kinase or diacylglycerol kinase. Stimulation by ATP depleted PIP2 from apical membranes, and PIP2 co‐ immunoprecipitated the β subunit of ENaC. ENaC was inhibited by ATP stimulation of P2Y2 receptors in Xenopus oocytes. Mutations in the PIP2 binding domain of βENaC but not γENaC reduced ENaC currents without affecting surface expression. Collectively, these data supply evidence for a novel and physiologically relevant regulation of ENaC in epithelial tissues. Although surface expression is controlled by its C terminus, N‐terminal binding of βENaC to PIP2 determines channel activity.

Integration‐Free Induced Pluripotent Stem Cells Model Genetic and Neural Developmental Features of Down Syndrome Etiology

Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of induced pluripotent stem cells (iPSCs) to model DS phenotypes, as a prototypical complex human disease, we generated bona fide DS and wild‐type (WT) nonviral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency and had remarkably similar lineage potency, differentiation kinetics, proliferation, and axon extension at early time points. However, at later time points DS cultures showed a twofold bias toward glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress‐induced apoptosis, and this could be prevented by the antioxidant N‐acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy 21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Nonviral DS iPSCs can therefore model features of complex human disease in vitro and provide a renewable and ethically unencumbered discovery platform. STEM CELLS2013;31:467–478

Control of ion transport in mammalian airways by protease activated receptors type 2 (PAR-2)

Protease‐activated receptors (PARs) are widely distributed in human airways. They couple to G‐proteins and are activated after proteolytic cleavage of the N terminus of the receptor. Evidence is growing that PAR subtype 2 plays a pivotal role in inflammatory airway diseases, such as allergic asthma or bronchitis. However, nothing is known about the effects of PAR‐2 on electrolyte transport in the native airways. PAR‐2 is expressed in airway epithelial cells, where they are activated by mast cell tryptase, neutrophil proteinase 3, or trypsin. Recent studies produced conflicting results about the functional consequence of PAR‐2 stimulation. Here we report that stimulation of PAR‐2 receptors in mouse and human airways leads to a change in electrolyte transport and a shift from absorption to secretion. Although PAR‐2 appears to be expressed on both sides of the epithelium, only basolateral stimulation results in inhibition of amiloride sensitive Na+ conductance and stimulation of both luminal Cl‐ channels and basolateral K+ channels. The present data indicate that these changes occur through activation of phospholipase C and increase in intracellular Ca2+, which activates basolateral SK4 K+ channels and luminal Ca2+‐dependent Cl‐ channels. In addition, the present data suggest a PAR‐2 mediated release of prostaglandin E2, which may contribute to the secretory response. In conclusion, these results provide further evidence for a role of PAR‐2 in inflammatory airway disease: stimulation of these receptors may cause accumulation of airway surface liquid, which, however, may help to flush noxious stimuli away from the affected airways.

Inhibition of Airway Na+ Transport by Respiratory Syncytial Virus

ABSTRACT In previous studies, we have shown that two major respiratory pathogens, influenza virus and parainfluenza virus, produce acute alterations in ion transport upon contacting the apical membrane of the respiratory epithelium. In the present study, we examine the effects on ion transport by the mouse tracheal epithelium of a third major respiratory pathogen, respiratory syncytial virus (RSV). RSV infections are associated with fluid accumulation in the respiratory tract and cause illnesses that range in severity from rhinitis, sinusitis, otitis media, and bronchitis to bronchiolitis and pneumonia. We find that within minutes of RSV contacting the apical membrane; it inhibits amiloride-sensitive Na+ transport by the epithelium. This effect is mediated by protein kinase C and is reproduced by recombinant viral F (fusion) protein. Since this inhibition is not accompanied by any alteration in the epithelial responses to carbachol or to forskolin plus 3-isobutyl-1-methylxanthine (IBMX), it is not due to a nonspecific toxic action of the virus. The inhibition also appears to require Toll-like receptor 4 and the presence of asialogangliosides in the apical membrane. Since the concentration range over which this inhibition is observed (102 to 105 PFU/ml) is comparable to the viral concentrations observed in clinical and experimental RSV infections, it seems likely that direct inhibition by the virus of epithelial Na+ transport may contribute to the fluid accumulation that is observed in RSV infections.

Effects of Purinergic Stimulation, CFTR and Osmotic Stress on Amiloride-sensitive Na+ Transport in Epithelia and Xenopus Oocytes

Both stimulation of purinergic receptors by ATP and activation of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibit amiloride-sensitive Na+ transport and activate Cl− secretion. These changes in ion transport may well affect cell volume. We therefore examined whether cell shrinkage or cell swelling do affect amiloride-sensitive Na+ transport in epithelial tissues or Xenopus oocytes and whether osmotic stress interferes with regulation of Na+ transport by ATP or CFTR. Stimulation of purinergic receptors by ATP/UTP or activation of CFTR by IBMX and forskolin inhibited amiloride-sensitive transport in mouse trachea and colon, respectively, by a mechanism that was Cl− dependent. When exposed to a hypertonic but not hypotonic bath solution, amiloride-sensitive Na+ transport was inhibited in mouse trachea and colon, independent of the extracellular Cl− concentration. Both inhibition of Na+ transport by hypertonic bath solution and ATP were additive. When coexpressed in Xenopus oocytes, activation of CFTR by IBMX and forskolin inhibited the epithelial Na+ channel (ENaC) in a Cl−dependent fashion. However, both hypertonic and hypotonic bath solutions showed only minor effects on amiloride-sensitive conductance, independent of the bath Cl− concentration. Moreover, CFTR-induced inhibition of ENaC could be detected in oocytes even after exposure to hypertonic or hypotonic bath solutions. We conclude that amiloride-sensitive Na+ absorption in mouse airways and colon is inhibited by cell shrinkage by a mechanism that does not interfere with purinergic and CFTR-mediated inhibition of ENaC.

Generation and characterization of leukemia inhibitory factor-dependent equine induced pluripotent stem cells from adult dermal fibroblasts

In this study we have reprogrammed dermal fibroblasts from an adult female horse into equine induced pluripotent stem cells (equiPSCs). These equiPSCs are dependent only on leukemia inhibitory factor (LIF), placing them in striking contrast to previously derived equiPSCs that have been shown to be co-dependent on both LIF and basic fibroblast growth factor (bFGF). These equiPSCs have a normal karyotype and have been maintained beyond 60 passages. They possess alkaline phosphatase activity and express eqNANOG, eqOCT4, and eqTERT mRNA. Immunocytochemistry confirmed that they produce NANOG, REX1, SSEA4, TRA1-60, and TRA1-81. While our equiPSCs are LIF dependent, bFGF co-stimulates their proliferation via the PI3K/AKT pathway. EquiPSCs lack expression of eqXIST and immunostaining for H3K27me3, suggesting that during reprogramming the inactive X chromosome has likely been reactivated to generate cells that have two active X chromosomes. EquiPSCs form embryoid bodies and in vitro teratomas that contain derivatives of all three germ layers. These LIF-dependent equiPSCs likely reflect a more naive state of pluripotency than equiPSCs that are co-dependent on both LIF and bFGF and so provide a novel resource for understanding pluripotency in the horse.

Control of epithelial ion transport by Cl- and PDZ proteins.

Inhibition of epithelial Na+ channels (ENaC) by the cystic fibrosis transmembrane conductance regulator (CFTR) has been demonstrated previously. Recent studies suggested a role of cytosolic Cl− for the interaction of CFTR with ENaC, when studied in Xenopus oocytes. In the present study we demonstrate that the Na+/H+-exchanger regulator factor (NHERF) controls expression of CFTR in mouse collecting duct cells. Inhibition of NHERF largely attenuates CFTR expression, which is paralleled by enhanced Ca2+-dependent Cl− secretion and augmented Na+ absorption by the ENaC. It is further demonstrated that epithelial Na+ absorption and ENaC are inhibited by cytosolic Cl− and that stimulation by secretagogues enhances the intracellular Cl− concentration. Thus, the data provide a clue to the question, how epithelial cells can operate as both absorptive and secretory units: Increase in intracellular Cl− during activation of secretion will inhibit ENaC and switch epithelial transport from salt absorption to Cl− secretion.

Mutations in Multiple Domains of c-Myb Disrupt Interaction with CBP/p300 and Abrogate Myeloid Transforming Ability

The c-myb proto-oncogene is a key regulator of hematopoietic cell proliferation and differentiation. MYB mRNA is expressed at high levels in, and is required for the proliferation of, most human myeloid and acute lymphoid leukemias. Recently, chromosomal translocation and genomic duplications of c-MYB have been identified in human T-cell acute leukemia. The present work focuses on the effects of mutations in different domains of the murine c-Myb protein on its transforming ability as defined by suppression of myelomonocytic differentiation and continued proliferation. Using both a novel myeloid cell line–based assay and a primary hematopoietic cell assay, we have shown that mutation of single residues in the transactivation domain important for CBP/p300 binding leads to complete loss of transforming ability. We also simultaneously mutated residues in the DNA-binding domain and the negative regulatory domain of the protein. These double mutants, but not the corresponding single mutants, show a complete loss of transforming activity. Surprisingly, these double mutants show severely impaired transactivation and are also defective for CBP/p300 binding. Our results imply that multiple Myb domains influence its interaction with CBP/p300, highlight the importance of this interaction for myeloid transformation, and suggest an approach for molecular targeting of Myb in leukemia. (Mol Cancer Res 2009;7(9):1477–86)

A new model to study neurodegeneration in ataxia oculomotor apraxia type 2

Ataxia oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive cerebellar ataxia. Recent evidence suggests that the protein defective in this syndrome, senataxin (SETX), functions in RNA processing to protect the integrity of the genome. To date, only patient-derived lymphoblastoid cells, fibroblasts and SETX knockdown cells were available to investigate AOA2. Recent disruption of the Setx gene in mice did not lead to neurobehavioral defects or neurodegeneration, making it difficult to study the etiology of AOA2. To develop a more relevant neuronal model to study neurodegeneration in AOA2, we derived neural progenitors from a patient with AOA2 and a control by induced pluripotent stem cell (iPSC) reprogramming of fibroblasts. AOA2 iPSC and neural progenitors exhibit increased levels of oxidative damage, DNA double-strand breaks, increased DNA damage-induced cell death and R-loop accumulation. Genome-wide expression and weighted gene co-expression network analysis in these neural progenitors identified both previously reported and novel affected genes and cellular pathways associated with senataxin dysfunction and the pathophysiology of AOA2, providing further insight into the role of senataxin in regulating gene expression on a genome-wide scale. These data show that iPSCs can be generated from patients with the autosomal recessive ataxia, AOA2, differentiated into neurons, and that both cell types recapitulate the AOA2 cellular phenotype. This represents a novel and appropriate model system to investigate neurodegeneration in this syndrome.

Effects of dietary lectins on ion transport in epithelia

Phytohemagglutinins are widely distributed in common food items. They constitute a heterogeneous group of proteins, which are often resistant to proteolysis in the gastrointestinal tract. Upon binding to the luminal membrane of intestinal cells, they can interfere with digestive, protective or secretory functions of the intestine. Phytohemagglutinins present in red kidney beans and jackbeans have been shown to induce diarrhea and hypersecretion in human airways, but the underlying mechanisms remain obscure. We examined how agglutinins from wheat germ (WGA), soy bean (SBA), red kidney beans (Pha‐E, Pha‐L), and jackbeans (Con‐A) affect ion transport in mouse airways and large intestine using Ussing chamber techniques. We found that Pha‐E, Pha‐L, and Con‐A but not WGA and SBA inhibit electrogenic Na+ absorption dose dependently in both colon and trachea. The inhibitory effects of Con‐A on Na+ absorption were suppressed by the sugar mannose, by inhibition of phospholipase C (PLC) and protein kinase C (PKC). Thus, nutritional phytohemagglutinins block salt absorption in a PLC‐ and PKC‐dependent manner, probably by inhibition of the epithelial Na+ channel (ENaC). This effect may be therapeutically useful in patients suffering from cystic fibrosis.