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.
The phorbol ester, phorbol 12-myristate 13-acetate (PMA), induces mucin secretion in the colonic tumor cell line T84 in a Ca(2+)-independent manner. To determine whether a specific protein kinase C (PKC) isoform is involved in colonic cells, we compared PMA-dependent mucin secretion by three human colonic tumor cell lines (T84, HT-29/A1, and LS 180) with the expression of PKC isoforms alpha, beta, delta, epsilon, and zeta, previously identified in human colon (L. A. Davidson, Y. H. Jiang, J. D. Derr, H. Aukema, J. R. Lupton, and R. S. Chapkin. Arch. Biochem. Biophys. 312:547-553, 1994). In each cell line PMA (10(-7) M) caused mucin secretion within 30 min. PMA-dependent mucin secretion was three to four times greater from HT-29/A1 and T84 cells than from LS 180 cells. All three-cell lines contained mRNA for PKC-alpha, PKC-epsilon, and PKC-zeta but not PKC-beta or -delta. Each cell line also expressed PKC-alpha, -epsilon, and -zeta protein. PKC-epsilon expression (mRNA and protein) was three to four times greater in HT-29/A1 and T84 cells than in LS 180 cells, correlating with PMA-responsive mucin secretion, whereas all cell lines contained similar levels of PKC-alpha mRNA and protein. When cells were stimulated by PMA, only PKC-epsilon was translocated from cytosol to membrane fractions early enough to stimulate mucin secretion. Because PKC-epsilon is also a Ca(2+)-independent isoform, it is likely to mediate mucin exocytosis in colonic cells.
We report a patient who presented with severe enterocolitis and apparent absence of Paneth, goblet, and enteroendocrine lineages from the small bowel and colon. The absorptive enterocyte seemed to be normal morphologically and functionally. Because normal enterocytes were present, we hypothesized that this patient had a developmental block in the differentiation of a common stem cell precursor for Paneth, goblet, and neuroendocrine lineages. By using antibodies to protein markers of each cell line, including some that are expressed early in the differentiation process, we aimed to study lineage development in this patient. From our data, we surmise that there may be a two-step process in lineage commitment. The stem cell may commit to an absorptive cell or a granule-containing cell. The intestinal crypts contain a population of multipotential stem cells from which all of the epithelial cell lineages are derived. On the basis of work in mice, a common progenitor cell located near the base of the crypt of Lieberkühn is thought to give rise to all four intestinal cell lines: absorptive enterocytes as well as Paneth, goblet, and enteroendocrine cells (1-4). Much work has been done to understand the differentiation and development from stem cells to the various terminally differentiated cell types of the intestinal epithelium and the factors that play a part in this process. Many factors have been implicated in this process, such as Wnt/-catenin signaling (5); Notch signaling, including the transcription factors Math1 (6) and Hes1 (7); homeobox transcription factors Cdx1 and Cdx2 (8); Kruppel-like factors KLF4 and KLF5 (9 -11); transcription factor Elf3 (12); platelet-derived growth factor (PDGF) A and its receptor, PDGF-Ra; the winged helix transcription factor Fkh6; the homeodomain transcription factor Nkx2-3; and Hox and ParaHox cluster genes, Sonic hedgehog, and bone morphogenetic proteins (13).Intestinal differentiation is difficult to study because of the technical challenge of growing nonmalignant epithelial cells in vitro. Although much has been learned from the use of normal, chimeric, and transgenic mice models, the process is still very ill defined. The work of Cheng and Leblond (2) on mouse models introduced the idea that the stem cell is the direct progenitor of each of the terminal lineages.We report a patient who presented with severe enterocolitis and apparent absence of Paneth, goblet, and enteroendocrine lineages from the small bowel and colon. The absorptive enterocyte seemed to be normal morphologically and functionally. Because normal enterocytes were present, we hypothesized that this patient had a developmental block in the differentiation of a common stem cell precursor for Paneth, goblet, and neuroendocrine lineages. These lineages have in common Received November 17, 2004; accepted February 3, 2005
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