Protein kinase C regulates transcription of the human guanylate cyclase C gene

Guanylate cyclase C is the receptor for the bacterial heat-stable enterotox ins and guanylin family of peptides, and mediates its action by elevating i ntracellular cGMP levels. Potentiation of ligand-stimulated activity of gua nylate cyclase C in human colonic T84 cells is observed following activatio n of protein kinase C as a result of direct phosphorylation of guanylate cy clase C. Here, we show that prolonged exposure of cells to phorbol esters r esults in a decrease in guanylate cyclase C content in il P-phorbol 12-myri state 13-acetate-treated cells, as a consequence of a decrease in guanylate cyclase C mRNA levels. The reduction in guanylate cyclase C mRNA was inhib ited when cells were treated with 4 beta -phorbol 12-myristate 13-acetate ( PMA) in the presence of staurosporine, indicating that a primary phosphoryl ation event by protein kinase C triggered the reduction in RNA levels. The reduction in guanylate cyclase C mRNA levels was not due to alterations in the half-life of guanylate cyclase C mRNA, but regulation occurred at the l evel of transcription of guanylate cyclase C mRNA. Expression in T84 cells of a guanylate cyclase C promoter-luciferase reporter plasmid, containing 1 973 bp of promoter sequence of the guanylate cyclase C gene, indicated that luciferase activity was reduced markedly on PMA treatment of cells, and th e protein kinase C-responsive element was present in a 129-bp region of the promoter, containing a HNF4 binding element. Electrophoretic mobility shif t assays using an oligonucleotide corresponding to the HNF4 binding site, i ndicated a decrease in binding of the factor to its cognate sequence in nuc lear extracts prepared from PMA-treated cells. We therefore show for the fi rst time that regulation of guanylate cyclase C activity can be controlled at the transcriptional level by cross-talk with signaling pathways that mod ulate protein kinase C activity. We also suggest a novel regulation of the HNF4 transcription factor by protein kinase C.