Local tissue stem cells have been described in airways of the lung but their contribution to normal epithelial maintenance is currently unknown. We therefore developed aggregation chimera mice and a whole-lung imaging method to determine the relative contributions of progenitor (Clara) and bronchiolar stem cells to epithelial maintenance and repair. In normal and moderately injured airways chimeric patches were small in size and not associated with previously described stem cell niches. This finding suggested that single, randomly distributed progenitor cells maintain normal epithelial homeostasis. In contrast we found that repair following severe lung injury resulted in the generation of rare, large clonal cell patches that were associated with stem cell niches. This study provides evidence that epithelial stem cells are dispensable for normal airway homeostasis. We also demonstrate that stem cell activation and robust clonal cellular expansion occur only during repair from severe lung injury.progenitor ͉ Clara cell ͉ BASC ͉ bronchiole ͉ repair

The current dopamine (DA) hypothesis of schizophrenia postulates striatal hyperdopaminergia and cortical hypodopaminergia. Although partial agonists at DA D2 receptors (D2Rs), like aripiprazole, were developed to simultaneously target both phenomena, they do not effectively improve cortical dysfunction. In this study, we investigate the potential for newly developed β-arrestin2 (βarr2)-biased D2R partial agonists to simultaneously target hyperand hypodopaminergia. Using neuron-specific βarr2-KO mice, we show that the antipsychotic-like effects of a βarr2-biased D2R ligand are driven through both striatal antagonism and cortical agonism of D2R-βarr2 signaling. Furthermore, βarr2-biased D2R agonism enhances firing of cortical fast-spiking interneurons. This enhanced cortical agonism of the biased ligand can be attributed to a lack of G-protein signaling and elevated expression of βarr2 and G proteincoupled receptor (GPCR) kinase 2 in the cortex versus the striatum. Therefore, we propose that βarr2-biased D2R ligands that exert region-selective actions could provide a path to develop more effective antipsychotic therapies.arrestin | antipsychotics | biased signaling | dopamine D2R | fast-spiking interneurons G protein-coupled receptors (GPCRs) represent the largest family of receptors in the human genome and are one of the most common targets of pharmaceutical drugs (1, 2). Upon ligand binding, GPCRs activate downstream G protein-dependent signaling pathways followed by phosphorylation of the receptor by G protein-coupled receptor kinases (GRKs) (3). Phosphorylation enhances association of the GPCR with β-arrestins (βarrs), and this combined process mediates desensitization of G-protein signaling (4) and internalization of GPCRs (5-7). Two isoforms of βarrs, βarr1 and βarr2, are widely coexpressed in most tissues in mammals and are 80% identical, but they can have either overlapping or distinct functions (8, 9). It is now firmly established that GPCRs activate downstream signaling pathways through not only canonical G-protein pathways but also, the ability of βarrs to scaffold distinct intracellular signaling complexes (10-12). Elucidation of these distinct G-protein and βarr signaling pathways has provided support for the concept of functional selectivity or biased signaling, wherein each signaling pathway has the ability to mediate distinct physiological responses (13). There are now several physiologically relevant examples of selective engagement of signaling pathways or selective GPCR ligands that target these different signaling pathways (13-15). Therefore, leveraging the concept of GPCR functional selectivity holds promise for the development of more selective therapeutic approaches. Dopamine (DA) is a catecholamine neurotransmitter that has been implicated in movement, reward, and cognition (16-19) as well as CNS disorders, such as schizophrenia, attention deficit hyperactivity disorder, Parkinson's disease, and obsessive-compulsive disorder (20-23). DA mediates its effects via GPCRs belonging to two major ...

Several studies in rodent models have shown that glycogen synthase kinase 3 β (GSK3β) plays an important role in the actions of antispychotics and mood stabilizers. Recently it was demonstrated that GSK3β through a β-arrestin2/protein kinase B (PKB or Akt)/protein phosphatase 2A (PP2A) signaling complex regulates dopamine (DA)-and lithium-sensitive behaviors and is required to mediate endophenotypes of mania and depression in rodents. We have previously shown that atypical antipsychotics antagonize DA D2 receptor (D2R)/β-arrestin2 interactions more efficaciously than G-protein-dependent signaling, whereas typical antipsychotics inhibit both pathways with similar efficacy. To elucidate the site of action of GSK3β in regulating DA-or lithium-sensitive behaviors, we generated conditional knockouts of GSK3β, where GSK3β was deleted in either DA D1-or D2-receptor-expressing neurons. We analyzed these mice for behaviors commonly used to test antipsychotic efficacy or behaviors that are sensitive to lithium treatment. Mice with deletion of GSK3β in D2 (D2GSK3β −/− ) but not D1 (D1GSK3β −/− ) neurons mimic antipsychotic action. However, haloperidol (HAL)-induced catalepsy was unchanged in either D2GSK3β −/− or D1GSK3β −/− mice compared with control mice. Interestingly, genetic stabilization of β-catenin, a downstream target of GSK3β, in D2 neurons did not affect any of the behaviors tested. Moreover, D2GSK3β −/− or D1GSK3β −/− mice showed similar responses to controls in the tail suspension test (TST) and dark-light emergence test, behaviors which were previously shown to be β-arrestin2-and GSK3β-dependent and sensitive to lithium treatment. Taken together these studies suggest that selective deletion of GSK3β but not stabilization of β-catenin in D2 neurons mimics antipsychotic action without affecting signaling pathways involved in catalepsy or certain mood-related behaviors.functional selectivity | schizophrenia | bipolar disorder | striatum | frontal cortex

Background: Expression of the G protein-coupled receptor LGR5 demarcates adult tissue stem cells in the intestine, stomach, hair follicle, and mammary epithelium. Results:LGR5 is rapidly and constitutively internalized to the trans-Golgi network at steady state. Conclusion: Internalization occurs through a potential phosphorylation domain within the C-terminal tail. Significance: An understanding of LGR5 trafficking dynamics is expected to clarify its role in signaling and stem cell biology.

Epithelial branching during the process of lung development results in the establishment of distinct functional zones, each of which is characterized by a unique cellular composition and repertoire of local progenitor cells. Significant new insights into cellular and molecular mechanisms of epithelial maintenance that provide insights into the pathophysiology of lung disease have been made in recent years. This review focuses on the complex structure-function relationship in the airway epithelium, how this epithelium is maintained in the normal state and repaired following injury, and how deregulation may contribute to airway disease and cancer.

Chronic lung diseases are marked by excessive inflammation and epithelial remodeling. Reduced Clara cell secretory function and corresponding decreases in the abundance of the major Clara cell secretory protein (CCSP) are characteristically seen in these disease states. We sought to define the impact of Clara cell and CCSP depletion on regulation of the lung inflammatory response. We used chemical and genetic mouse models of Clara cell and CCSP deficiency (CCSP(-/-)) coupled with Pseudomonas aeruginosa LPS elicited inflammation. Exposure of Clara cell-depleted or CCSP(-/-) mice to LPS resulted in augmented inflammation as assessed by polymorphonuclear leukocyte recruitment to the airspace. Gene expression analysis and pathway modeling of the CCSP(-/-) inflammatory response implicated increased TNF-alpha signaling. Consistent with this model was the demonstration of significantly elevated TNF-alpha in airway fluid of LPS-stimulated CCSP(-/-) mice compared with similarly exposed wild-type mice. Increased LPS-elicited TNF-alpha production was also observed in cultured lung macrophages from CCSP(-/-) mice compared with wild-type mice. We demonstrate that macrophages from Clara cell-depleted and CCSP(-/-) mice displayed increased Toll-like receptor 4 surface expression. Our results provide evidence that Clara cells can attenuate inflammation through regulation of macrophage behavior, and suggest that epithelial remodeling leading to reduced Clara cell secretory function is an important factor that increases the intensity of lung inflammation in chronic lung disease.

SummaryWnt-b-catenin signaling regulates cell fate during organ development and postnatal tissue maintenance, but its contribution to specification of distinct lung epithelial lineages is still unclear. To address this question, we used a Cre recombinase (Cre)-LoxP approach to activate canonical Wnt signaling ectopically in developing lung endoderm. We found that persistent activation of canonical Wnt signaling within distal lung endoderm was permissive for normal development of alveolar epithelium, yet led to the loss of developing bronchiolar epithelium and ectasis of distal conducting airways. Activation of canonical Wnt led to ectopic expression of a lymphoid-enhancing factor and a T-cell factor (LEF and TCF, respectively) and absence of SRY (sex-determining region Y)-box 2 (SOX2) and tumor protein p63 (p63) expression in proximal derivatives. Conditional loss of SOX2 in airways phenocopied epithelial differentiation defects observed with ectopic activation of canonical Wnt. Our data suggest that Wnt negatively regulates a SOX2-dependent signaling program required for developmental progression of the bronchiolar lineage.

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