Figure 1.
Cortisol exerts its permissive action at the plasma membrane level.
A, a schematic record of the tension curve. Histamine and isoprenaline were used to contract and relax the tracheal spiral, respectively. Each of the potentiators (including cortisol) was added to this system when contraction reached the plateau level. B, the relaxation effect of isoprenaline at different concentration of BSA-cortisol. The concentration gradient ranged from 0, 840, 1680 to 2520 ng/ml. The way to calculate therapeutic index and normalization were described in Materials and methods. * p<0.05 when 2520 ng/ml cortisol group is compared with baseline group (n=4 in each group).
Figure 2.
The expression prolife of EMT.
A, immunohistochemical staining showed the expression of EMT in chondrocytes of tracheal cartilage, but not in airway smooth muscles. B, immunofluorescent labeling of EMT on chondrocytes (upper three subpanels), coronary smooth muscle cells (middle three subpanels) and airway smooth muscle cells (lower three subpanels). Specificity of EMT-immunolabeling was documented by antibody preabsorption (insets in upper and middle subpanels). The nuclei were stained blue with DAPI, and EMT was stained green by the immunofluoresence method. Scale bar: 20 μm. C, comparison of EMT expression in human airway smooth muscle (ASM) cells and vascular smooth muscle (VSM) cells. Data were extracted from open-access microarray datasets (GEO accession GSE2883, GSE13168). The expression values were normalized to GAPDH or ACTA2 in each dataset, ** p<0.001. D, the circadian transcription patterns of EMT and Per3. Note that Per3 (a typical circadian gene) showed circadian fluctuation in transcription, while EMT transcription did not show circadian fluctuation. Data were extracted from an open-access microarray dataset (GEO accession GSE 11922).
Figure 3.
EMT inhibition potentiates the spasmolytic action of isoprenaline.
A, famotidine, a potent EMT inhibitor, when added in the replacement of cortisol, achieved a more profound permissive effect for isoprenaline (n=3 in each group), * p<0.05 vs. control. B, there was no additive effect when famotidine and cortisol were administrated in sequential order. C, with famotidine preincubation, cortisol did not further potentiate the spasmolytic action of isoprenaline. D, a similar contractile response was produced by histamine with or without famotidine pretreatment. E, SPG method to quantify catecholamine uptake by tracheal chondrocyte. Pretreatment with EMT inhibitors (cortisol or famotidine) hampered catecholamine uptake (50 μM norepinephrine, 20 min incubation). Fluorescent intensity was measured in approximately 50 cells for each group, * p<0.05, ** p<0.001 vs. control. a.u., arbitrary unit.
Figure 4.
Protein kinase C (PKC) phosphorylates EMT and induces EMT internalization.
A, okadaic acid, an inhibitor of protein phosphatase PP1/2A, partially imitated the permissive action of cortisol (n=3 in each group), * p<0.05 vs. control. B, bioinformatics analyses of guinea pig EMT amino acid sequence revealed threonine 302 and 391 as consensus PKC site, which were also conserved across species. C, an overall view of predicated guinea pig EMT, showing transmembrane (TM) helices in orange. Other residues were represented in green. Two consensus PKC site, one located at juxtamembrane region (Thr302) and the other in TM helix were depicted in blue (upper and middle insets). A typical caveolin-binding motif (CBM) was also shown in silver (lower inset). D, endocytic inhibitor filipin III (FIII), but not concanavalin A (ConA) blocked the permissive effect induced by PKC activator, PMA (n=4 in each group). E, immunofluorescent staining of EMT in vascular smooth muscle cells with different pharmacological manipulation (upper three subpanels, control; middle three subpanels, PMA treatment; lower three subpanels, filipin III exposure and then PMA treatment). The nuclei were stained blue with DAPI, and EMT was stained green by the immunofluoresence method. Scale bar: 20 μm.
Figure 5.
A putative EMT-centered model explains the permissive action of cortisol in trachea.
EMT as the direct target is inhibited by cortisol, and this action slows down the reuptake process of catecholamines by chondrocytes, leading to extracellular catecholamines accumulation and more considerable adrenergic signaling activation in smooth muscle cells. Moreover, cortisol-initiated membrane signaling triggers PKC activation, which phosphorylates EMT and results in EMT internalization. Signaling components associated with airway disorders in genetic studies are labeled with asterisks (*).