Phorbol 12-Myristate 13-Acetate Up-regulates the Transcription of MUC2 Intestinal Mucin via Ras, ERK, and NF-kappa B

Department of Medicine, University of California, San Francisco, San Francisco, California, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2002; 277(36):32624-31. DOI: 10.1074/jbc.M200353200
Source: PubMed


MUC2 is a secretory mucin normally expressed by goblet cells of the intestinal epithelium. It is overexpressed in mucinous type colorectal cancers but down-regulated in colorectal adenocarcinoma. Phorbol 12-myristate 13-acetate (PMA) treatment of colon cancer cell lines increases MUC2 expression, so we have undertaken a detailed analysis of the effects of PMA on the promoter activity of the 5'-flanking region of the MUC2 gene using stably and transiently transfected promoter reporter vectors. Protein kinase C inhibitors (bisindolylmaleimide, calphostin C) and inhibitors of mitogen-activated protein/extracellular signal regulated kinase kinase (MEK) (PD98059 and U0126) suppressed up-regulation of MUC2. Src tyrosine kinase inhibitor PP2, a protein kinase A inhibitor (KT5720), and a p38 inhibitor (SB 203580) did not affect transcription. Western blotting and reverse transcription-PCR analysis confirmed these results. In addition, co-transfections with mutants of Ras, Raf, and MEK showed that the induction of MUC2 promoter activity by PMA required these three signaling proteins. Our results demonstrate that PMA activates protein kinase C, stimulating MAP kinase through a Ras- and Raf-dependent mechanism. An important role for nuclear factor kappaB (NF-kappaB) was also demonstrated using the inhibitor caffeic acid phenethyl ester and electrophoretic mobility shift assays. Such identification of pathways involved in MUC2 up-regulation by PMA in the HM3 colon cancer cell line may serve as a model for the effects of cytokines and growth factors, which regulate MUC2 expression during the progression of colorectal cancer.

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    • "This correlation is consistent with the molecular observation that p-ERK activates c-Fos transcription by interacting on its SRE with ETS-domain family proteins, such as Elk-1 (Galbraith and Espinosa, 2011). To further test this link, we treated cells with tetradecanoylphorbol acetate (TPA), which strongly activates c-Fos via the ERK pathway (Lee et al., 2002). We found that TPA treatment yielded prolonged periods of p-ERK presence in the nucleus as well as continued activation of c-Fos (see Supplemental Results and Figure S1M). "
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    ABSTRACT: Transcription is a stochastic process occurring mostly in episodic bursts. Although the local chromatin environment is known to influence the bursting behavior on long timescales, the impact of transcription factors (TFs)-especially in rapidly inducible systems-is largely unknown. Using fluorescence in situ hybridization and computational models, we quantified the transcriptional activity of the proto-oncogene c-Fos with single mRNA accuracy at individual endogenous alleles. We showed that, during MAPK induction, the TF concentration modulates the burst frequency of c-Fos, whereas other bursting parameters remain mostly unchanged. By using synthetic TFs with TALE DNA-binding domains, we systematically altered different aspects of these bursts. Specifically, we linked the polymerase initiation frequency to the strength of the transactivation domain and the burst duration to the TF lifetime on the promoter. Our results show how TFs and promoter binding domains collectively act to regulate different bursting parameters, offering a vast, evolutionarily tunable regulatory range for individual genes.
    Cell Reports 06/2014; 8(1). DOI:10.1016/j.celrep.2014.05.053 · 8.36 Impact Factor
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    • "Among the inflammatory mediators, PMA can substitute for diacylglycerol, and is known to activate PKC, which in turn initiates signaling cascades that lead to a variety of cell events [18-20]. In the induction of mucins, several studies have also reported that PMA stimulates the expression of MUC5AC and MUC5B in the airway and intestinal epithelial cells [18, 21, 22]. "
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    ABSTRACT: Phorbol 12-myristate 13-acetate (PMA) is widely used as a protein kinase C (PKC) activator, PKC is involved in the secretion of mucins. MUC16, one of the membrane-bound mucins, is produced in human airway epithelial cells. However, the effect and signaling pathway of PMA on MUC16 expression in human airway epithelial cells has not been reported. Therefore, the effect and brief signaling pathway of PMA on MUC16 expression were investigated in human airway epithelial cells in this study. In the mucin-producing human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells, the effect and signaling pathway of PMA on MUC16 expression were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA) for p38 mitogen-activated protein kinase (MAPK). PMA increased MUC16 expression, and activated phosphorylation of p38 MAPK. However, it did not activate phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). SB203580 (p38 MAPK inhibitor) inhibited PMA-induced MUC16 expression, while U0126 (ERK1/2 inhibitor) did not. In addition, the knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked PMA-induced MUC16 mRNA expression. Rottlerin (PKCδ inhibitor) inhibited PMA-induced MUC16 expression, and also inhibited the phosphorylation of activated p38 MAPK by PMA. These results show for the first time that PMA-induced MUC16 expression is regulated by activation of the PKCδ and p38 MAPK signaling pathway in human airway epithelial cells.
    Clinical and Experimental Otorhinolaryngology 09/2012; 5(3):161-9. DOI:10.3342/ceo.2012.5.3.161 · 0.85 Impact Factor
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    • "HM3 (human colon epithelial), HeLa (human cervix epithelial), A549 (Human lung epithelial), and HMEEC-1 (human middle ear epithelial) cells were maintained and used as described previously [5], [6], [74], [76], [77], [97], [98]. HM3 and HMEEC-1 cells are from Dr. Y.S. Kim and Dr. David Lim, respectively, as described previously [40], [71], [99], [100]. HeLa and A549 cells are from ATCC. "
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    ABSTRACT: CARD-containing MAGUK protein 1 (CARMA1) plays a crucial role in regulating adaptive immune responses upon T-cell receptor (TCR) activation in T cells. Its role in regulating host mucosal innate immune response such as upregulation of mucin remains unknown. Here we show that CARMA1 acts as a key signaling mediator for synergistic upregulation of MUC5AC mucin by bacterium nontypeable Haemophilus influenzae (NTHi) and phorbol ester PMA in respiratory epithelial cells. NTHi-induced TLR-dependent TRAF6-MKK3-p38 MAPK signaling pathway synergizes with PKCθ-MEK-ERK signaling pathway. CARMA1 plays a crucial role in mediating this synergistic effect via TRAF6, thereby resulting in synergistic upregulation of MUC5AC mucin. Thus our study unveils a novel role for CARMA1 in mediating host mucosal innate immune response.
    PLoS ONE 01/2012; 7(1):e31049. DOI:10.1371/journal.pone.0031049 · 3.23 Impact Factor
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