Glucocorticoids and Myosin5b loss-of-function induce heightened PKA signaling in addition to membrane traffic defects.

Loss-of-function mutations in the non-conventional myosin Vb (Myo5b) result in Microvillus Inclusion Disease (MVID), and massive secretory diarrhea that often begins at birth. Myo5b mutations disrupt the apical recycling endosome (ARE) and membrane traffic, resulting in reduced surface expression of apical membrane proteins. ARE disruption also results in constitutive PDK1 gain-of-function. In MVID, decreased surface expression of apical anion channels involved in Cl- extrusion, such as CFTR, should reduce fluid secretion into the intestinal lumen. But the opposite phenotype is observed. To explain this contradiction and the onset of diarrhea, we hypothesized that signaling effects downstream of Myo5b loss-of-function synergize with higher levels of glucocorticoids to activate PKA and CFTR. Data from intestinal cell lines, human MVID, and Myo5b KO mouse intestine revealed changes subcellular redistribution of PKA activity to the apical pole, increased CFTR phosphorylation, and establishment of apical cAMP gradients in Myo5b-defective cells exposed to physiological levels of glucocorticoids. These cells also displayed net secretory fluid fluxes and transepithelial currents mainly from PKA-dependent Cl- secretion. We conclude that Myo5b defects result in PKA stimulation that activate residual channels on the surface when intestinal epithelia are exposed to glucocorticoids at birth. [Media: see text] [Media: see text].