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

Abstract

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.

Fig. 1

Confocal microscopy of airway epithelial and smooth muscle cell cultured cells used for transcription factor expression studies. Cells were prepared as described in Methods. Morphologic characteristics (panels A, D) and signals from cytokeratin-FITC (panels C, F) are consistent with cell-type, with airway epithelial cells (AEC) but not airway smooth muscle cells (ASM) expected to stain positive. The signals in panels B, Eare from the fluorescent nuclear counterstain DAPI. Magnification = 20X.

Fig. 2

Confocal microscopy of airway epithelial and smooth muscle cell cultured cells used for transcription factor expression studies. Cells were prepared as described in Methods. Morphologic characteristics (panels A, D) and signals from vimentin-Cy3 (panels C, F) are consistent with cell-type, with smooth muscle, but not epithelial cells, expected to stain positive. The signals in panels B, Eare from the fluorescent nuclear counterstain DAPI. Magnification = 20X.

Fig. 3

Global expression of transcription factors in airway epithelial and smooth muscle cells based on a query of >300 factors. Shown is the number of transcription factors detected by consensus oligonucleotide binding under any of the four culture conditions (see Methods). Factors expressed only in one cell-type are denoted as unique, and when detected in both as common.

Fig. 4

Conditional changes in transcription factor expression/binding in airway epithelial and smooth muscle cells. Cells were treated with the β-agonist isoproterenol, a pro-asthma cocktail (see Methods), or both for 24 hrs, and nuclear extracts prepared. The y -axis represents the number of transcription factors that changed by condition, with the number of upregulated factors in the black bars and the number of downregulated factors in the white bars.

Fig. 5

Regulation of transcription factors that are expressed in both airway epithelial and smooth muscle cells. Shown are the transcription factors where expression was common to both cell-types under at least one of the four conditions. The red bars indicated an increase and the blue bars a decrease in expression/binding by the indicated condition. T he yellow bars represent the scenario where expression was detectable, but there was no change with the condition. White bars indicate that there was no detectable expression for the transcription factor under the given condition.