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MUCOSAL BIOLOGY

Chronic PKC-₂ activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane CFTR targeting

¹Department of Integrative Biology and Department of Pharmacology and Physiology and ²Division of Gastroenterology, Hepatology, and Nutrition, Department of Internal Medicine, University of Texas Health Science Center Medical School, Houston, Texas

Submitted 28 July 2005 ; accepted in final form 16 March 2006

We investigated the effects of chronically applied PKC-stimulating phorbol esters on subcellular CFTR expression and localization in polarized HT-29 Cl.19A monolayers. Modulation of PKC activity with the PKC--specific agonist 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) or nonisoform-selective PMA altered monolayer CFTR immunofluorescence. A decrease in the CFTR signal within the luminal cellular pole was noted with both phorbol esters. Volumetric analysis of the intracellular CFTR signal revealed that both compounds promoted CFTR accumulation into punctate vesicle-like structures found adjacent to the cellular tight junction [labeled with zona occludens (ZO)-1 antibody], extending basally (DOPPA) into the cell. Puncta were more frequent with DOPPA and larger in size with PMA. DOPPA also promoted ZO-1 accumulation at tricellular corners associated with enhanced CFTR puncta number. The observed loss of CFTR immunofluorescence signal induced by low-dose PMA was related to CFTR sequestration into fewer cytoplasmic puncta and correlated with larger increases in PKC substrate phosphorylation. Both phorbol esters downregulated steady-state cellular CFTR mRNA levels by 70%. However, the effects of DOPPA and PMA were largely independent of CFTR biosynthesis: expression levels were 80–85% of control, and the glycosylation status of immunoprecipitated protein remained largely unchanged. Thus changes in cellular CFTR localization correlated with our companion study showing that PMA-induced inhibition of transcellular cAMP-dependent short-circuit current (I_SC) was accompanied by cytoplasmic PKC-₂ accumulation and modest activation of PKC-₁ and PKC-. The inhibitory effect of DOPPA on I_SC was related solely to increased cytoplasmic PKC-₂ levels. Thus PKC-₂ is hypothesized to participate in the regulation of CFTR apical plasma membrane targeting within the constitutive cellular biosynthetic pathway.

protein kinase C; immunofluorescence; short-circuit current; cystic fibrosis transmembrane conductance regulator; phorbol ester