Submitted on July 28, 2005
Accepted on March 16, 2006
Chronic PKC
activation in HT-29 Cl.19A colonocytes (2) Prevents cAMP-mediated ion secretion by inhibiting apical membrane CFTR targeting
James R Broughman1, Limin Sun1, Shahid Umar1, Joseph H Sellin1, and Andrew P Morris1*
1 Integrative Biology, Pharmacology and Physiology, University of Texas Health Science Center at Houston, Houston, Texas, United States
* To whom correspondence should be addressed. E-mail: andrew.p.morris{at}uth.tmc.edu.
We investigated the effects of chronically applied, protein kinase C (PKC1) stimulating phorbol esters on cystic fibrosis transmembrane conductance regulator (CFTR) expression and localization in polarized HT29 Cl.19A monolayers. Modulation of PKC activity with the PKC
-specific agonist 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) or non-isoform selective phorbol 12-myristate 13-acetate (PMA) altered monolayer CFTR immunofluorescence (IMF). CFTR signal decreased within the luminal cellular pole 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, extending basally into the cell with DOPPA. Puncta were more numerous with DOPPA, but larger with PMA. DOPPA also promoted ZO-1 accumulation at tri-cellular corners associated with enhanced puncta number. The observed loss of CFTR IMF signal induced by PMA was related to CFTR sequestration into cytoplasmic puncta, and correlated with larger increases in PKC substrate phosphorylation. Both phorbol esters down-regulated steady-state cellular CFTR mRNA levels by 70%. These effects were independent of CFTR biosynthesis: expression levels were 80-85% of control and glycosylation remained largely unchanged. Thus, changes in cellular CFTR localization correlate with data presented in our companion paper showing that PMA induced inhibition of transcellular (Tc) ISC-cAMP was accompanied by the cytoplasmic PKC2 accumulation, and modest activation of PKC1 and PKC
isoforms. The inhibitory effect of DOPPA on ISC was related solely to increased cytoplasmic PKC 2 levels. PKC2 is hypothesized to participate in the regulation of apical CFTR targeting within the constitutive cellular biosynthetic pathway.
