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

Abstract

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 MUC2expression, so we have undertaken a detailed analysis of the effects of PMA on the promoter activity of the 5′-flanking region of theMUC2 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 ofMUC2 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 κB (NF-κB) 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 regulateMUC2 expression during the progression of colorectal cancer. 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 MUC2expression, so we have undertaken a detailed analysis of the effects of PMA on the promoter activity of the 5′-flanking region of theMUC2 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 ofMUC2 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 κB (NF-κB) 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 regulateMUC2 expression during the progression of colorectal cancer. phorbol 12-myristate 13-acetate protein kinase C mitogen-activated protein kinase protein kinase A extracellular signal-regulated kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase nuclear factor κB caffeic acid phenethyl ester reverse transcription electrophoretic mobility shift assays epidermal growth factor c-Jun N-terminal kinase Mucins are very large proteins featuringO-glycosylated, tandemly repeated serine- and threonine-rich regions. They are synthesized by the epithelial cells lining the gastrointestinal, respiratory, and genitourinary tracts. Genomic and cDNA sequencing has identified at least fifteen different mucin genes, which encode either secretory or membrane-associated proteins (1Moniaux N. Escande F. Porchet N. Aubert J.P. Batra S.K. Front. Biosci. 2001; 6: D1192-D1206Crossref PubMed Google Scholar, 2Williams S.J. Wreschner D.H. Tran M. Eyre H.J. Sutherland G.R. McGuckin M.A. J. Biol. Chem. 2001; 276: 18327-18336Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 3Yin B.W. Lloyd K.O. J. Biol. Chem. 2001; 276: 27371-27375Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar). Mucins are expressed in a characteristic tissue- and cell type-specific manner. MUC2 is one of four structurally related but differentially expressed secretory mucins located on chromosome 11p15. Within the intestinal epithelium, MUC2 is highly expressed in goblet cells but absent from the absorptive cell type (4Ho S.B. Niehans G.A. Lyftogt C. Yan P.S. Cherwitz D.L. Gum E.T. Dahiya R. Kim Y.S. Cancer Res. 1993; 53: 641-651PubMed Google Scholar, 5Audie J.P. Janin A. Porchet N. Copin M.C. Gosselin B. Aubert J.P. J. Histochem. Cytochem. 1993; 41: 1479-1485Crossref PubMed Scopus (410) Google Scholar, 6Chang S.-Y. Dohrman A.F. Basbaum C., Ho, S.B. Tsuda T. Toribara N.W. Gum J.R. Kim Y.S. Gastroenterology. 1994; 107: 28-36Abstract Full Text PDF PubMed Google Scholar, 7Sylvester P.A. Myerscough N. Warren B.F. Carlstedt I. Corfield A.P Durdey P. Thomas M.G. J. Pathol. 2001; 195: 327-335Crossref PubMed Scopus (58) Google Scholar). Altered expression of mucin genes occurs in many epithelial cancers. Specifically, low expression of MUC2 has been reported in colorectal adenocarcinoma, whereas a very high level of MUC2 expression is observed in mucinous colorectal carcinomas, a distinct histological type of colorectal cancer (4Ho S.B. Niehans G.A. Lyftogt C. Yan P.S. Cherwitz D.L. Gum E.T. Dahiya R. Kim Y.S. Cancer Res. 1993; 53: 641-651PubMed Google Scholar, 5Audie J.P. Janin A. Porchet N. Copin M.C. Gosselin B. Aubert J.P. J. Histochem. Cytochem. 1993; 41: 1479-1485Crossref PubMed Scopus (410) Google Scholar, 7Sylvester P.A. Myerscough N. Warren B.F. Carlstedt I. Corfield A.P Durdey P. Thomas M.G. J. Pathol. 2001; 195: 327-335Crossref PubMed Scopus (58) Google Scholar, 8Hanski C. Riede E. Gratchev A. Foss H.D. Bohm C. Klussmann E. Hummel M. Mann B. Buhr H.J. Stein H. Kim Y.S. Gum J. Riecken E.O. Lab. Invest. 1997; 77: 685-695PubMed Google Scholar). However, relatively little is known about the mechanisms responsible for regulation ofMUC2 gene expression in vivo. Phorbol esters such as PMA1function as tumor promoters and have been reported to modulate diverse cellular responses such as gene transcription, cellular growth and differentiation, programmed cell death, the immune response, and receptor desensitization through protein kinase C (PKC) signaling pathways. 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 (9Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2365) Google Scholar, 10Jaken S. Curr. Opin. Cell Biol. 1996; 8: 168-173Crossref PubMed Scopus (407) Google Scholar, 11Parekh D.B. Ziegler W. Parker P.J. EMBO J. 2000; 19: 496-503Crossref PubMed Scopus (512) Google Scholar). Phorbol esters, as well as cytokines and bacterial lipopolysaccharides, have been shown to up-regulate mucin genes (12Velcich A. Augenlicht L. J. Biol. Chem. 1993; 268: 13956-13961Abstract Full Text PDF PubMed Google Scholar, 13Hong D.-H. Petrovics G. Anderson W.B. Forstner J. Forstner G. Am. J. Physiol. 1999; 277: G1041-G1047PubMed Google Scholar, 14Levine S.J. Larivee C.L. Logun C.W. Angus C.S. Ognibene F.P. Shelhamer J.H. Am. J. Respir. Cell Mol. Biol. 1995; 12: 196-204Crossref PubMed Scopus (204) Google Scholar, 15Yoon J.-H. Gray T. Guzman K. Koo J.S. Nettesheim P. Am. J. Respir. Cell Mol. Biol. 1997; 16: 724-731Crossref PubMed Scopus (88) Google Scholar, 16Koo J.S. Jetten A.M. Belloni P. Yoon J.-H. Kim Y.-D. Nettesheim P. Biochem. J. 1999; 338: 351-357Crossref PubMed Google Scholar, 17Basbaum C. Lemjabbar H. Longphre M., Li, D. Gensch E. McNamara N. Am. J. Respir. Crit. Care Med. 1999; 160: S44-S48Crossref PubMed Scopus (119) Google Scholar). We recently reported that PMA up-regulates several mucin genes, including MUC2, in colon cancer cell lines (18Han S.Y. Lee M.S. Kim H.R. Baek S.H. Ahn D.H. Chae H.S. Erickson R.H. Sleisenger M.H. Kim Y.S. Int. J. Oncol. 2000; 17: 487-494PubMed Google Scholar). However, detailed analysis of the downstream signaling pathways involved in PMA/PKC-induced up-regulation ofMUC2 has not been done. One well studied mode of PKC-mediated signaling involves transmission of signals from PKC to mitogen-activated protein kinases (MAPKs). MAPK activation by PMA has been reported to occur via both Ras-dependent and Ras-independent pathways: PC-12 rat adrenal pheochromocytoma (19Thomas S.M. DeMarco M. D'Arcangelo G. Halegoua S. Brugge J.S. Cell. 1992; 68: 1031-1040Abstract Full Text PDF PubMed Scopus (503) Google Scholar, 20Wood K.W. Sarnecki C. Roberts T.M. Blenis J. Cell. 1992; 68: 1041-1050Abstract Full Text PDF PubMed Scopus (662) Google Scholar), Jurkat leukemic T cells (21Li Y.Q., Hi, C.S.T. Costabile M. Goh D. Der C.J. Ferrante A. J. Immunol. 1999; 162: 3316-3320PubMed Google Scholar), and primary rat ventricular myocytes (22Chiloeches A. Paterson H.F. Marais R. Clerk A. Marshall C.J. Sugen P.H. J. Biol. Chem. 1999; 274: 19762-19770Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) have exhibited Ras-dependent activation of MAPK by PMA; but in NIH3T3 mouse myeloma cells (23deVries-Smits A.M. Burgering B.M. Leevers S.J. Marshall C.J. Bos J.L. Nature. 1992; 357: 602-604Crossref PubMed Scopus (304) Google Scholar), COS-1 (24Howe L.R. Leevers S.J. Gomez M. Kakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (631) Google Scholar), and 293 embryonal kidney cell lines (25Ming X.F. Burgering B.M.T. Wennstrom S. Claesson-Welsh L. Heldin C.H. Bos J.L. Kozma S.C. Thomas G. Nature. 1994; 371: 426-429Crossref PubMed Scopus (204) Google Scholar), this activation appears to be Ras-independent. Thus, the involvement of Ras in signaling processes initiated by PMA appears to be cell type- dependent and specifically determined by which signaling pathways have been activated and/or the cell's repertoire of kinases. HM3 human colon cancer cell line contains the most common K-rasmutation type found in colorectal cancers, the glycine to aspartate mutation at codon 12. Because many growth factors and cytokines utilize Ras-dependent signaling pathways, this cell line serves as a model system for studying alterations in gene expression that occur in the progression of colorectal cancers. In this study we show that PMA stimulates expression of theMUC2 gene in HM3 cells through activation of PKC. SuchMUC2 up-regulation is Ras- and Raf-dependent, requires activation of the MEK/ERK signaling pathway, and ultimately involves activation of a nuclear factor, NF-κB. TriReagent and PMA were obtained from Sigma. Bisindolylmaleimide I, calphostin C, KT5720, PD98059, U0126, CAPE, AG126, PP2, PP3, and SB203580 were purchased from Calbiochem. Antibodies for ERK1/2 (p44/42 MAPK), SAPK/JNK, and p38 were purchased from New England Biolabs/Cell Signaling. Secondary antibodies were purchased from Zymed Laboratories Inc., South San Francisco, CA. Oligonucleotides were synthesized by Operon, Alameda, CA. HM3, a subclone of the LS174T adenocarcinoma cell line, was maintained at 37 °C in 5% CO2 atmosphere in Dulbecco’s modified Eagle’s minimum medium containing 10% heat-inactivated fetal bovine serum with penicillin and streptomycin. HM3 cells stably transfected with MUC2 promoter (−2864/+19) pGL2 (Promega) luciferase construct (HM3M2) were maintained in medium containing 600 μg/ml G148 (Geneticin). The HM3M2 cells were serum-starved overnight and then pretreated with inhibitors for 1 h before exposure to 0.25 μm PMA for 4 C was used a of located the was using TriReagent and was with and reverse using in a of One of this was in a using with the of 5% for MUC2 were and was as using a of from The was at °C for by at °C for °C for and °C for and for were used to this as were on and were using were using the from The expression for and activated were a from was a from for type and were a from of San MUC2 promoter reporter assays pGL2 (Promega) containing of the MUC2 gene 5′-flanking J.R. Kim Y.S. Biochem. J. 1997; PubMed Scopus Google Scholar, W. M. Kim J.-H. Gum J. Kim Y. Basbaum C. PubMed Scopus Google Scholar). In addition, the MUC2 promoter region of the kinase promoter in a luciferase expression was used to the of the to transcription from a promoter W. M. Kim J.-H. Gum J. Kim Y. Basbaum C. PubMed Scopus Google Scholar). were transfected in with of of and of luciferase as to for 1 the cells were serum-starved overnight in fetal bovine serum and then with PMA or as in the The cells were and luciferase were using (Promega) with the luciferase activity were with to luciferase activity to for in using stably transfected HM3M2 cells were with overnight serum 1 h of inhibitor and then 4 h of PMA activity was using the from is as the of four from a with by and determined by with in the cell were in μg/ml μg/ml 1 and The protein of was determined by using the Protein of protein were to 10% and to J. T. A Scholar), which was with bovine serum in with with primary with containing and then with antibodies to were by using the were to Res. 19: PubMed Scopus Google Protein were determined using the A to the promoter region from to was by and reverse and with and kinase England of the were also W. M. Kim J.-H. Gum J. Kim Y. Basbaum C. PubMed Scopus Google Scholar). proteins were with for in a containing of in at and before on a in of protein to was demonstrated by of a of antibodies for or of were with nuclear for on PMA MUC2 in a and with the at 4 h analysis that PMA a in 4 h at μm In addition, PMA activity of the MUC2 luciferase reporter in HM3M2 cells in a to μm promoter showed increases in activity treatment with PMA reported J.R. Kim Y.S. Biochem. J. 1997; PubMed Scopus Google Scholar), containing of were The construct was for as it exhibited the level of PMA Bisindolylmaleimide is a highly PKC inhibitor that as a inhibitor for the of PKC. μm PMA induction of MUC2 promoter activity by inhibitor of PKC, calphostin C, also promoter activation by was used to that and calphostin C also a in endogenous MUC2 It be that in these a of the effects of PMA and inhibitors to the relatively high of MUC2 in from the of theMUC2 J.R. Kim Y.S. Biochem. J. 1997; PubMed Scopus Google Scholar). KT5720, a inhibitor of did not the up-regulation of activity or kinase inhibitor on MUC2 promoter PP3, a for PP2, and epidermal growth factor receptor kinase inhibitor also to Thus, PKC inhibitors were to the up-regulation of MUC2 promoter that this kinase is responsible for the role of we used and to the activity of inhibitors MUC2 induction by PMA and the level of endogenous MUC2 using a highly and inhibitor of p38 MAP to theMUC2 promoter activity by PMA AG126, inhibitor of tyrosine also MUC2 up-regulation in a Because is a downstream of ERK1/2 was for to MUC2 promoter activation was shown are of four The results obtained using MAP kinase inhibitors were confirmed with Western using antibodies to of and that both and were of PMA p38 was shown to be in this cell line and by PMA observed in SAPK/JNK, which occurs as to of and but this was from cells as a on p38 or but with MEK and activity of the MUC2 the with MEK activated the in the of and this activation was not by the of PMA co-transfections with MUC2 induction in a MUC2 promoter reporter co-transfections using did not affect with or of the both activation by PMA and promoter activity as a in MUC2 transcription to observed PMA HM3 cells transfected with were also with a was observed CAPE, inhibitor of the induction of MUC2 by PMA was confirmed using to in endogenous In nuclear from HM3 cells to of the transcription factor to the However, nuclear from cells a protein that to the and was by the of of a mutation the that to to the the of the of the transcription of the this to of to have little in this but in a in the of the was observed of the a it appears that the is involved up-regulation by but involvement of the to be to to a A luciferase reporter containing the kinase promoter downstream from the of MUC2 was activated by PMA that the in this region as of MUC2 transcription. In this we determined that PMA in HM3 The activity of a MUC2 construct stably transfected HM3 human colon cancer cell line HM3M2 was by that up-regulation occurs at the We also determined that PMA treatment was to up-regulate transcription from containing the identified The promoter were observed for and The construct was for of high activity and it determined to be the and the J.R. Kim Y.S. Biochem. J. 1997; PubMed Scopus Google W. M. Kim J.-H. Gum J. Kim Y. Basbaum C. PubMed Scopus Google Scholar). the signaling pathways involved in the effects of inhibitors and expression on transcription were A is shown in PKC inhibitors and calphostin C up-regulation by that PKC activation by phorbol ester is responsible for the induction ofMUC2 promoter The induction of by PMA was not suppressed by either inhibitor or by Src type tyrosine kinase inhibitor The up-regulation by both and receptor kinase inhibitor are with of of by W. Biochem. J. 2000; PubMed Scopus Google Scholar). is used as a for the of to up-regulation also that receptor via of not The of involvement of Src type tyrosine kinases is in to results obtained using the cell line, which showed that MUC2 up-regulation was that PKC of the for of PKC-mediated activation of the signaling has been well but the and of to activation to be by several and to the by Ras W. Biochem. J. 2000; PubMed Scopus Google Scholar). is a membrane-associated with at codon a Ras protein with a high for The is to as a inhibitor of Ras activation through of the required for from the Mol. Cell. Biol. 8: PubMed Scopus Google Scholar, R. Y. C. Paterson H. Marshall C.J. PubMed Scopus Google Scholar). The of to activation has been reported to be cell (19Thomas S.M. DeMarco M. D'Arcangelo G. Halegoua S. Brugge J.S. Cell. 1992; 68: 1031-1040Abstract Full Text PDF PubMed Scopus (503) Google Scholar, 20Wood K.W. Sarnecki C. Roberts T.M. Blenis J. 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In addition, very MUC2 activity by in a that Raf, with Ras, in MUC2 activation by PMA and that both are Ras is to a of signaling pathways, including the MAP kinase pathway, the pathway, a pathway, a pathway, and a W. Biochem. J. 2000; PubMed Scopus Google Scholar, C. S. Physiol. 1999; PubMed Scopus Google Scholar, K. A. A. Der C.J. Cell Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A. C. 1999; PubMed Google Scholar). downstream from in to which MAP kinase pathways were involved in MUC2 kinase inhibitors were for to MUC2 transcription. inhibitors of MEK U0126) as well as MUC2. the involvement of MEK in the effects of we and of MEK with the promoter construct and showed that PMA induction ofMUC2 activity was by that the induction ofMUC2 activity by PMA is AG126, which is inhibitor of a Western using antibodies for of and showed that ERK1/2 is in to PMA We were not to demonstrate PMA on p38 was shown to be activated in this cell line, and it showed a in by PMA treatment in However, the SB203580 inhibitor of p38 did not that p38 is not required for of MUC2 transcription. is important it has been shown that p38 is a downstream kinase responsible for Ras activation of J.L. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). of MUC2 transcription by that is activation of Ras and downstream which may from the that HM3 cells of is the most common found in colorectal carcinomas, the studied The has been reported to be the has and to the and F. J. Pathol. 1999; PubMed Scopus Google Scholar). It has also been reported that but not Ras the of very low in the and not on factors for However, the of to MUC2 transcription that the of activity F. J. Pathol. 1999; PubMed Scopus Google Scholar), is to by The of the mutation in HM3 cells may be to a activation of the and the MUC2 mutants of both and as well as MEK inhibitors and as well as with activation of the for this is the of of ERK1/2 in Western of from HM3 with activated up-regulation of MUC2 transcription. The of PMA to up-regulate MUC2 transcription in the of that may be activation of the distinct and or that is of pathways that are not activated by different including transcription factors and have been identified as downstream of including kinase and as well as transcription factors such as and signal and activator of transcription proteins W. Biochem. J. 2000; PubMed Scopus Google Scholar, C. S. Physiol. 1999; PubMed Scopus Google Scholar). was shown to MUC2 transcription by W. M. Kim J.-H. Gum J. Kim Y. Basbaum C. PubMed Scopus Google Scholar), we were to demonstrate the on transcription. that downstream of ERK1/2 may be different for Thus, whereas was to activation W. M. Kim J.-H. Gum J. Kim Y. Basbaum C. PubMed Scopus Google Scholar), it appears that is not responsible for activation of NF-κB. are required to and this has been reported to M. A. Biochem. Res. 1999; PubMed Scopus Google Scholar). A was identified in the region of the MUC2 promoter and J.R. Kim Y.S. Biochem. J. 1997; PubMed Scopus Google Scholar). In analysis of the of PMA on MUC2 this region also appears to PMA well as a of proteins also to this a in J.R. Kim Y.S. Biochem. J. 1997; PubMed Scopus Google Scholar). However, using this we were to and D.-H. S. C. Li, J. R. C. B. N. D. E. S.-Y. B. H. M. H. and Y. S. results. It is that this region of the promoter is important but that up-regulation involves activity of or nuclear factors that are to this region of the MUC2 are required to the MUC2 promoter factor a role in the regulation of genes involved in the and of cell of is through and of inhibitor the to to the and genes M. Y. Immunol. 2000; PubMed Scopus Google Scholar). The of for the kinase and are in the of The activity of is also by at on both The kinases responsible for these may Ras, and PKC. and the of at are M. Y. Immunol. 2000; PubMed Scopus Google Scholar, F. A.M. Curr. Opin. Cell Biol. 1999; PubMed Scopus Google Scholar). MUC2 was shown to be by via in epithelial cells W. M. Kim J.-H. Gum J. Kim Y. Basbaum C. PubMed Scopus Google Scholar). CAPE, inhibitor of endogenous as well as MUC2 transcription, that is also involved in MUC2 transcription by using containing the identified MUC2 demonstrated that nuclear from HM3 cells nuclear from the of the was demonstrated using containing the region of theMUC2 promoter from to of the kinase Because was not shown to be involved in MUC2 is activated via pathway, such as or In we demonstrate that treatment of HM3 cells with PMA results in activation of PKC, stimulating the MAP kinase and MUC2 in a Ras- and Raf-dependent manner. was also shown to be involved in MUC2 up-regulation by In to on MUC2 transcription using as the effects of PMA are of Src and and are by the involvement of the MUC2 We H. Erickson and for and for and and for