Environmental controls on fungal community composition and abundance over 3 years in native and degraded shrublands.

Soil fungal communities have high local diversity and turnover, but the relative contribution of environmental and regional drivers to those patterns remains poorly understood. Local factors that contribute to fungal diversity include soil properties and the plant community, but there is also evidence for regional dispersal limitation in some fungal communities. We used different plant communities with different soil conditions and experimental manipulations of both vegetation and dispersal to distinguish among these factors. Specifically, we compared native shrublands with former native shrublands that had been disturbed or converted to pasture, resulting in soils progressively more enriched in carbon and nutrients. We tested the role of vegetation via active removal, and we manipulated dispersal by adding living soil inoculum from undisturbed native sites. Soil fungi were tracked for 3 years, with samples taken at ten time points from June 2006 to June 2009. We found that soil fungal abundance, richness, and community composition responded primarily to soil properties, which in this case were a legacy of plant community degradation. In contrast, dispersal had no effect on soil fungi. Temporal variation in soil fungi was partly related to drought status, yet it was much broader in native sites compared to pastures, suggesting some buffering due to the increased soil resources in the pasture sites. The persistence of soil fungal communities over 3 years in this study suggests that soil properties can act as a strong local environmental filter. Largely persistent soil fungal communities also indicate the potential for strong biotic resistance and soil legacies, which presents a challenge for both the prediction of how fungi respond to environmental change and our ability to manipulate fungi in efforts such as ecosystem restoration.

Crosstalk between Gi and Gq/Gs pathways in airway smooth muscle regulates bronchial contractility and relaxation.

Receptor-mediated airway smooth muscle (ASM) contraction via G(alphaq), and relaxation via G(alphas), underlie the bronchospastic features of asthma and its treatment. Asthma models show increased ASM G(alphai) expression, considered the basis for the proasthmatic phenotypes of enhanced bronchial hyperreactivity to contraction mediated by M(3)-muscarinic receptors and diminished relaxation mediated by beta(2)-adrenergic receptors (beta(2)ARs). A causal effect between G(i) expression and phenotype has not been established, nor have mechanisms whereby G(i) modulates G(q)/G(s) signaling. To delineate isolated effects of altered G(i), transgenic mice were generated overexpressing G(alphai2) or a G(alphai2) peptide inhibitor in ASM. Unexpectedly, G(alphai2) overexpression decreased contractility to methacholine, while G(alphai2) inhibition enhanced contraction. These opposite phenotypes resulted from different crosstalk loci within the G(q) signaling network: decreased phospholipase C and increased PKCalpha, respectively. G(alphai2) overexpression decreased beta(2)AR-mediated airway relaxation, while G(alphai2) inhibition increased this response, consistent with physiologically relevant coupling of this receptor to both G(s) and G(i). IL-13 transgenic mice (a model of asthma), which developed increased ASM G(alphai), displayed marked increases in airway hyperresponsiveness when G(alphai) function was inhibited. Increased G(alphai) in asthma is therefore a double-edged sword: a compensatory event mitigating against bronchial hyperreactivity, but a mechanism that evokes beta-agonist resistance. By selective intervention within these multipronged signaling modules, advantageous G(s)/G(q) activities could provide new asthma therapies.

Allele-specific binding of airway nuclear extracts to polymorphic beta2-adrenergic receptor 5' sequence.

Like other intronless G protein-coupled receptor genes, the beta(2)-adrenergic receptor (beta(2)AR) has minimal genetic space for population variability, and has attained such via multiple coding and noncoding polymorphisms. Yet most clinical studies use the two nonsynonymous polymorphisms of the coding region for association analysis despite low levels of linkage disequilibrium with some promoter and 5'UTR polymorphisms. To assess the potential for allele-specific transcription factor binding to beta(2)AR 5'-flanking sequence, 3'-biotin-labeled oligonucleotide duplexes were synthesized. Each was centered on variable sites representing major or minor alleles found in the human population with frequencies of 5% or greater (20 polymorphic sites). Electrophoretic mobility shift assays were performed using human airway smooth muscle or airway epithelial cell nuclear extracts. Many of these polymorphisms resulted in an alteration in binding, and both major allele and minor allele dominance were observed. For example, in airway smooth muscle nuclear extracts, 10 polymorphisms decreased and 2 increased binding, whereas 5 showed no differences. Concordance between airway smooth muscle and epithelial cell nuclear extract binding to polymorphic alleles was found in only approximately 50% of cases. There was no tendency for the rare variants to be more likely to have altered nuclear extract binding compared to the more common variants. Taken together, these results provide potential mechanisms by which beta(2)AR 5'-flanking polymorphisms affect obstructive lung phenotypes.

Heterogeneity of transcription factor expression and regulation in human airway epithelial and smooth muscle cells.

Transcription factors represent a major mechanism by which cells establish basal and conditional expression of proteins, the latter potentially being adaptive or maladaptive in disease. The complement of transcription factors in two major structural cells of the lung relevant to asthma, airway epithelial and smooth muscle cells, is not known. A plate-based platform using nuclear extracts from these cells was used to assess potential expression by binding to oligonucleotide consensus sequences representing >300 transcription factors. Four conditions were studied: basal, beta-agonist exposure, culture under proasthmatic conditions (IL-13, IL-4, TGF-beta, and leukotriene D(4)), and the dual setting of beta-agonist with proasthmatic culture. Airway epithelial cells expressed 70 transcription factors, whereas airway smooth muscle expressed 110. High levels of multiple transcription factors not previously recognized as being expressed in these cells were identified. Moreover, expression/ binding patterns under these conditions revealed extreme discordance in the direction and magnitude of change between the cell types. Singular (one cell type displayed regulation) and antithetic (both cell types underwent expression changes but in opposite directions) regulation dominated these patterns, with concomitant regulation in both cell types being rare (<10%). beta-Agonist evoked up- and downregulation of transcription factors, which was highly influenced by the proasthmatic condition, with little overlap of factors regulated by beta-agonists under both conditions. Together, these results reveal complex, cell type-dependent networks of transcription factors in human airway epithelium and smooth muscle that are dynamically regulated in unique ways by beta-agonists and inflammation. These factors may represent additional components in asthma pathophysiology or potential new drug targets.

Alpha2A- and alpha2C-adrenergic receptors form homo- and heterodimers: the heterodimeric state impairs agonist-promoted GRK phosphorylation and beta-arrestin recruitment.

Dimerization of seven transmembrane-spanning receptors diversifies their pharmacologic and physiologic properties. The alpha(2)-adrenergic receptor (alpha(2)AR) subtypes A and C are both expressed on presynaptic nerves and act to inhibit norepinephrine release via negative feedback. However, in vivo and in vitro studies examining the roles of the two individual alpha(2A)- and alpha(2C)AR subtypes are not readily reconciled. We tested the hypothesis that the receptors form homo- and heterodimers and that the alpha(2A)-alpha(2C) heterodimer has unique properties. SDS-PAGE of epitope-tagged receptors revealed potential oligomers including dimers. BRET of live HEK-293 cells transfected with the subtypes fused to Rluc or YFP revealed that both subtypes form dimers and the heterodimer. A lower BRET(50) for the alpha(2A)-alpha(2C) heterodimer (0.79 +/- 0.20) compared to that of the alpha(2A) or alpha(2C) homodimer (2.331 +/- 0.44 or 3.67 +/- 0.69, respectively) suggests that when both subtypes are expressed, there is a greater likelihood that the two receptors will form the heterodimer than homodimers. Co-immunoprecipitation studies confirmed homo- and heterodimer formation. The presence of the alpha(2C)AR within the heterodimer resulted in a marked reduction in the level of GRK2-mediated alpha(2A)AR phosphorylation, which was accompanied by a qualitative attenuation of beta-arrestin recruitment. Signaling of the alpha(2A)-alpha(2C) heterodimer to the beta-arrestin-dependent activation of Akt was decreased compared to that of the alpha(2A)AR homodimer, while p44/p42 MAP kinase activation was unaffected. Thus, the alpha(2C)AR alters alpha(2A)AR signaling by forming oligomers, and these complexes, which appear to be preferred over the homodimers, should be considered a functional signaling unit in cells in which both subtypes are expressed.