Entamoeba histolytica Cysteine Proteinase 5 Evokes Mucin Exocytosis from Colonic Goblet Cells via αvβ3 Integrin
Fig 5
PKCδ is activated downstream of PI3K and is necessary for EhCP5 induced mucin secretion.
A. Mucins from LS174T goblet cells metabolically labeled with 3H-glucosamine were treated with the broad spectrum PKC inhibitor bisindolylmaleimide I (10μM), infected with Eh for 2 h and assayed for 3H-labeled mucin secretion. A panel of PKC inhibitors including Rottlerin (20μM), Bisindolylmaleimide IX (10μM) and Gö6983 (10μM) was used to discern the critical isoform in response to WTEh secreted components (50μg/mL). B. WTEh secreted components (50μg/mL) and recombinant EhCP5 (5μg) were added to LS174T cells and activation of PI3K and PKC determined over a 15-minute period. C. Confocal microscopy on fixed monolayers infected with WTEh and EhCP5- were imaged with a Phospho-PKCδ antibody to detect activation. The dotted white line indicates the position of Eh. Arrows indicate regions of interest where PKCδ is phosphorylated at the Eh-goblet cell contact site. Scale bar 10 μM. D. Quantification of confocal images was performed by measuring the concentration of phosphorylated PKCδ within the host cell in the immediate contact site by Eh. E. LS174T cells were pretreated with either the PI3K inhibitor wortmannin or PKC inhibitor bisindolylmaleimide I and stimulated with WTEh secreted components over a 15 minute period. *** p <0.001, **p <0.01.