Induction of mucin gene expression in human colonic cell lines by PMA is dependent on PKC-ε

Abstract

Treatment of HT-29 cells with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), induces MUC2 expression. To investigate the role of PKC in regulating mucin genes in intestinal cells, we examined the regulation of MUC1, MUC2, MUC5AC, MUC5B, and MUC6 expression in two human mucin-producing colonic cell lines, T84 and HT29/A1. T84 and HT29/A1 cells (at 80-90% confluency) were exposed to 100 nM PMA for 0, 3, and 6 h. Twofold or greater increases in mRNA levels for MUC2 and MUC5AC were observed in both cell lines during this time period, whereas the levels of MUC1, MUC5B, and MUC6 mRNAs were only marginally affected. These results indicated that PKC differentially regulates mucin gene expression and that it may be responsible for altered mucin expression. Our previous results suggested that the Ca(2+)-independent PKC-epsilon isoform appeared to mediate PMA-regulated mucin exocytosis in these cell lines. To determine if PKC-epsilon was also involved in MUC2/MUC5AC gene induction, HT29/A1 cells were stably transfected with either a wild-type PKC-epsilon or a dominant-negative ATP-binding mutant of PKC-epsilon (PKC-epsilon K437R). Overexpression of the dominant-negative PKC-epsilon K437R blocked induction of both mucin genes, whereas PMA-induced mucin gene expression was not prevented by overexpression of wild-type PKC-epsilon. PMA-dependent MUC2 mucin secretion was also blocked in cells overexpressing the dominant-negative PKC-epsilon K437R. On the basis of these observations, PKC-epsilon appears to mediate the expression of two major gastrointestinal mucins in response to PMA as well as PMA-regulated mucin exocytosis.

Phorbol 12-Myristate 13-Acetate Up-regulates the Transcription of MUC2Intestinal Mucin via Ras, ERK, and NF-κB

“…PMA can substitute for diacylglycerol, the endogenous activator of PKC, and it has been used as a model agent to study the mechanisms utilized by growth factors, hormones, and cytokines to regulate growth and differentiation of cells (9 -11). Phorbol esters, as well as cytokines and bacterial lipopolysaccharides, have been shown to up-regulate mucin genes (12)(13)(14)(15)(16)(17). We recently reported that PMA up-regulates several mucin genes, including MUC2, in colon cancer cell lines (18).…”

nPKCε, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells

“…For instance, overexpression of nPKCε caused increased secretion of prolactin from GH4 cells (5) and of insulin from pancreatic ␤ cells (26). Conversely, overexpression of a dominant negative nPKCε mutant or downregulation of nPKCε using siRNAs diminished or abolished stimulated secretion from HT29 cells (25), pancreatic ␤ cells (42), lacrimal gland cells (28), and chromaffin cells (26). Other PKC isoforms have been implicated in regulated secretion from other cells [e.g., cPKC␤ (9,37)], but the reports generally are neither as rigorous in approach, as numerous, nor as consistently positive in their identification as for nPKCε.…”

“…Even though such a correlation is consistent with causality, alone it offers an insufficient level of proof. Additionally, only cPKC␤I and nPKCε have been implicated strongly in the regulation of exocytotic secretion (5,9,25,26,28,37,42), whereas nPKC␦ has been associated primarily with cell differentiation and apoptosis (27,57). Hence, in the present study we have pursued the identity of the PKC isoform active in regulated mucin secretion, using pharmacological, molecular, and genetic manipulations to alter PKC isoform activity or expression levels.…”

Cell Signaling through the Protein Kinases cAMP-dependent Protein Kinase, Protein Kinase Cϵ, and RAF-1 Regulates Amphotropic Murine Leukemia Virus Envelope Protein-induced Syncytium Formation

“…The PKC␣-, PKC␤-, PKC␦-, and PKC⑀-overexpressing NIH3T3 cell lines were generated as described previously utilizing the ⑀MTH vector (17). NIH3T3/DN-PKC⑀ cells overexpressing the kinase-inactive mutant of PKC⑀ were constructed by converting lysine 437 within the catalytic domain to an arginine and then cloned into the epitope-tagging vector p⑀MTH, as described previously (24). NIH3T3/BXB Raf cells overexpressing the N-terminally truncated, activated Raf-1 were produced by cloning into the p⑀MTH vector (25).…”

“…This mutant was constructed with an amino acid substitution at Lys-437 in the catalytic domain to prevent ATP binding (24). As presented in Table III, A-MuLV envelope protein-induced cell-cell fusion was observed in NIH3T3 cells treated with H89 to inhibit PKA activity.…”