Phorbol 12-Myristate 13-Acetate Up-regulates the Transcription of
MUC2 Intestinal Mucin via Ras, ERK, and NF-?B*
Received for publication, January 11, 2002, and in revised form, June 4, 2002
Published, JBC Papers in Press, June 20, 2002, DOI 10.1074/jbc.M200353200
Hae-Wan Lee,a–cDae-Ho Ahn,a,b,dSuzanne C. Crawley,a,eJian-Dong Li,fJames R. Gum, Jr.,a,e
Carol B. Basbaum,gNancy Q. Fan,hDavid E. Szymkowski,hSang-Young Han,a,iBong H. Lee,a,j
Marvin H. Sleisenger,a,eand Young S. Kim,a,e,k
From theaGastrointestinal Research Laboratory, Veterans Affairs Medical Center, San Francisco, California 94121, the
Departments ofeMedicine andgAnatomy, University of California, San Francisco, California 94143, thefGonda
Department of Cell and Molecular Biology, House Ear Institute, and the Department of Otolaryngology, University of
Southern California, Los Angeles, California 90057, andhRoche Bioscience, Palo Alto, California 94304
MUC2 is a secretory mucin normally expressed by
goblet cells of the intestinal epithelium. It is overex-
pressed in mucinous type colorectal cancers but down-
regulated in colorectal adenocarcinoma. Phorbol 12-my-
ristate 13-acetate (PMA) treatment of colon cancer cell
lines increases MUC2 expression, so we have under-
taken 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 (bisin-
dolylmaleimide, calphostin C) and inhibitors of mito-
gen-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. West-
ern 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 ?B (NF-?B) 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.
Mucins are very large proteins featuring O-glycosylated, tan-
demly repeated serine- and threonine-rich regions. They are
synthesized by the epithelial cells lining the gastrointestinal,
respiratory, and genitourinary tracts. Genomic and cDNA se-
quencing has identified at least fifteen different mucin genes,
which encode either secretory or membrane-associated pro-
teins (1–3). 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 (4–7). 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 (4, 5, 7, 8). However, relatively little is known about the
mechanisms responsible for regulation of MUC2 gene expres-
sion 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 path-
ways. 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 li-
popolysaccharides, have been shown to up-regulate mucin
genes (12–17). We recently reported that PMA up-regulates
several mucin genes, including MUC2, in colon cancer cell lines
(18). However, detailed analysis of the downstream signaling
pathways involved in PMA/PKC-induced up-regulation of
MUC2 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 activa-
tion by PMA has been reported to occur via both Ras-dependent
and Ras-independent pathways: PC-12 rat adrenal pheochro-
mocytoma (19, 20), Jurkat leukemic T cells (21), and primary
rat ventricular myocytes (22) have exhibited Ras-dependent
* This work was supported by the Department of Veterans Affairs
Medical Research Service, United States Public Health Service Grant
CA 24321 from the National Institutes of Health and the Theodora Betz
Foundation Fund. The costs of publication of this article were defrayed
in part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
bBoth authors contributed equally to this work.
cPresent address: Dept. of Surgery, Chunchon Sacred Heart Hospi-
tal, Chunchon, Korea 200-060.
dPresent address: Dept. of Surgery, College of Medicine, Pundang
CHA Hospital, Kyonggi-do, 463-712, Korea.
iPresent address: Dept. of Medicine, Dong-A University College of
Medicine, Pusan, Korea 602-715.
jPresent address: Dept. of General Surgery, Hallym University,
Chunchon, Korea 431-070.
kTo whom correspondence should be addressed: Gastrointestinal Re-
search Laboratory (151M2), Veterans Affairs Medical Center, 4150
Clement St., San Francisco, CA 94121. Tel.: 415-750-2095; Fax: 415-
750-6972; E-mail: email@example.com.
1The abbreviations used are: PMA, phorbol 12-myristate 13-acetate;
PKC, protein kinase C; MAPK, mitogen-activated protein kinase; PKA,
protein kinase A; ERK, extracellular signal-regulated kinase; MEK,
mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase; NF-?B, nuclear factor ?B; CAPE, caffeic acid phenethyl ester;
RT, reverse transcription; EMSA, electrophoretic mobility shift assays;
EGF, epidermal growth factor; JNK, c-Jun N-terminal kinase.
THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 277, No. 36, Issue of September 6, pp. 32624–32631, 2002
Printed in U.S.A.
This paper is available on line at http://www.jbc.org
by guest on December 26, 2015
activation of MAPK by PMA; but in NIH3T3 mouse myeloma
cells (23), COS-1 (24), and 293 embryonal kidney cell lines (25),
this activation appears to be Ras-independent. Thus, the in-
volvement of Ras in signaling processes initiated by PMA ap-
pears 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-ras mutation 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 the MUC2 gene in HM3 cells through activation of PKC.
Such MUC2 up-regulation is Ras- and Raf-dependent, requires
activation of the MEK/ERK signaling pathway, and ultimately
involves activation of a nuclear factor, NF-?B.
Materials—TriReagent and PMA were obtained from Sigma. Bisin-
dolylmaleimide 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 pur-
chased 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.
Tissue Culture—HM3, a subclone of the LS174T adenocarcinoma cell
line, was maintained at 37 °C in 5% CO2atmosphere 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) lucif-
erase construct (HM3M2) were maintained in medium containing 600
?g/ml G148 (Geneticin).
Inhibitor Assays—The HM3M2 cells were serum-starved overnight
and then pretreated with inhibitors for 1 h before exposure to 0.25 ?M
PMA for 4 h. Calphostin C was used under a fluorescent lamp of 15
watts located 15 cm above the plates.
RNA Isolation and RT-PCR—Total RNA was isolated using TriRe-
agent (Molecular Research Center Inc.), and 3 ?g was primed with
random hexamers and reverse transcribed using Superscript II (In-
vitrogen) in a final volume of 50 ?l. One microliter of this mixture was
PCR-amplified in a 10-?l reaction using AmpliTaq DNA polymerase
(Applied Biosystems) with the addition of 5% dimethyl sulfoxide. Prim-
ers for MUC2 were (forward) 5?-TGC CTG GCC CTG TCT TTG-3? and
(reverse) 5?-CAG CTC CAG CAT GAG TGC-3?. 18 S rRNA was simul-
taneously amplified as an internal standard, using a 9:1 ratio of 3?
blocked/unblocked (alternate) primers from Ambion QuantumRNATM
18 S Internal Standards kit. The PCR reaction mixture was denatured
at 94 °C for 5 min followed by 30 cycles at 94 °C for 30 s, 60 °C for 30 s,
and 72 °C for 30 s. Alternatively, blocked and unblocked primers for
?-actin (forward: 5?-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG
C-3?; reverse: 5?-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT
G-3?) were used to amplify this message as an internal control. All PCR
products were separated on ethidium bromide-stained gels, and band
intensities were integrated using NIH Image software.
Plasmids, Transient Transfection, and Luciferase Reporter Assays—
Plasmids were prepared using the Plasmid MAXI Prep Kit from Qia-
gen. The expression vectors for Ha-Ras, dominant-negative N17Ras,
and constitutively activated v-Ha-Ras were a gift from Geoffrey Cooper
(Boston University, Boston, MA). HMEK1(K97R) was a gift from Alan
Saltiel (Parke-Davis Pharmaceutical Research Division, Ann Arbor,
MI). Expression vectors for wild type and dominant-negative pp90rsk
(pp90rsk?C) were a gift from Warner Greene, University of California
San Francisco. MUC2 promoter reporter assays employed pGL2 vector
(Promega) containing various regions of the MUC2 gene 5?-flanking
sequence described previously (26, 27). In addition, the MUC2 promoter
region ?1528/?1307, subcloned upstream of the thymidylate kinase
promoter in a luciferase expression vector TK-32, was used to evaluate
the ability of the NF-?B site to enhance transcription from a minimal
promoter (27). Cells were typically transfected in 12-well plates with 8.0
?l of Superfect (Qiagen), plus 2 ?g of test plasmid, and 0.1 ?g of pRLO
(a promoterless Renilla luciferase vector; Promega) as an internal con-
trol to correct for transfection efficiency. After 1 day the cells were
serum-starved overnight in 1.25% fetal bovine serum and then treated
with PMA or vehicle as indicated in the figure legends. The cells were
harvested and luciferase activities were measured using dual Lucifer-
aseTMReporter Assay System (Promega) with the Monolight 2010 Lu-
minometer (Analytical Luminescent Laboratory). Firefly luciferase ac-
tivity measurements were normalized with respect to pRLO Renilla
luciferase activity to correct for variations in transfection efficiency.
Inhibitor experiments using stably transfected HM3M2 cells were car-
ried out similarly with overnight serum starvation, 1 h of inhibitor
pretreatment, and then 4 h of PMA treatment. Promoter activity was
assessed using the Luciferase Assay Kit from Promega. Data is pre-
sented graphically as the average of four replicates from a representa-
tive experiment with standard deviation provided by error bars and
statistical significance determined by Student’s t test with confidence
levels indicated in the figure legends.
Western Blotting—After various treatments, total cell lysates were
prepared in 10 mM Tris-HCl, pH 6.8, 0.4 mM EDTA, 2% SDS, 10 ?g/ml
leupeptin, 10 ?g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10
mM sodium fluoride, 0.4 mM sodium orthovanadate, and 10 mM pyro-
phosphate. The protein concentration of supernatant was determined
by using the bicinchoninic acid-based BCA Protein Assay Kit (Pierce).
Equal amounts of protein were subjected to 10% SDS-polyacrylamide
gel electrophoresis and transferred to nitrocellulose (28), which was
blocked with 3% bovine serum albumin in Tris-buffered saline (TBS; 10
mM Tris-HCl with 150 mM NaCl, pH 7.4), probed with specific primary
antibodies, washed with TBS containing 0.1% Tween, and then probed
with secondary antibodies conjugated to horseradish peroxidase. Im-
munoreactive bands were visualized by chemiluminescence using the
Renaissance kit (PerkinElmer Life Sciences).
Electrophoretic Mobility Shift Assays (EMSA)—Nuclear extracts
were prepared according to Ref. 29. Protein concentrations were deter-
mined using the Bradford Assay method (Bio-Rad). A double-stranded
oligonucleotide probe corresponding to the human MUC2 promoter
region from ?1458 to ?1430 (5?-CGTCCTTGGGTTTCCCCAGGGCTA-
GTGC-3?) was prepared by annealing forward and reverse primers and
labeled with [?-32P]ATP and T4 polynucleotide kinase (New England
Biolabs). Mutated versions of the same probe were also used, where
MT3 ? 5?-CGTCCTTGGGTTGAATAAGGGCTAGTGC-3? (27). Nuclear
proteins were incubated with radiolabeled probe for 20 min in a solution
containing 3 ?g of poly(dI-dC) in 10 mM HEPES-KOH at pH 7.9, 210 mM
NaCl, 0.75 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, and 12.5% glycerol before separation on
a 4% (19:1 acrylamide/bisacrylamide) polyacrylamide gel in Tris ace-
tate-EDTA buffer. Specificity of protein binding to radiolabeled oligonu-
cleotides was demonstrated by addition of a 100-fold excess of unla-
belled competing oligonucleotide. For supershift assays, antibodies
specific for p50, p65, or c-Rel subunits of NF-?B (Santa Cruz Biotech-
nology) were preincubated with nuclear extracts for 30 min on ice.
PMA Up-regulates MUC2 mRNA Level and Induces MUC2
Transcriptional Activity—PMA increased MUC2 mRNA in a
time- and dose-dependent manner with the peak effect at 4 h
(Fig. 1A). Densitometric analysis revealed that PMA caused a
4-fold increase in mRNA levels after 4 h at 0.5 ?M concentration
(Fig. 1B). In addition, PMA up-regulated transcriptional activ-
ity of the MUC2 luciferase reporter in HM3M2 cells in a dose-
dependent manner up to 0.5 ?M (Fig. 1C).
PMA Increases the Transcriptional Activity of MUC2 Promoter
Reporter Constructs—Transiently transfected MUC2 promoter
deletion constructs all showed significant (p ? 0.001) increases in
activity upon treatment with PMA (Fig. 2). As reported previ-
ously (26), constructs containing sequence up-stream of base
?1308 were significantly more active than smaller constructs.
The ?1628/?19 construct was chosen for further experiments as
it exhibited the highest level of PMA responsiveness.
PKC but Not PKA Mediates the Activation of MUC2 Promoter
Activity by PMA—Bisindolylmaleimide I is a highly selective
cell-permeable PKC inhibitor that acts as a competitive inhib-
itor for the ATP-binding site of PKC. 0.1 ?M bisindolylmaleim-
ide inhibited PMA induction of MUC2 promoter activity by 80%
(Fig. 3A). Another specific inhibitor of PKC, calphostin C, also
inhibited PMA-induced MUC2 promoter activation by ?80%.
RT-PCR was used to confirm that bisindolylmaleimide and
calphostin C also caused a reduction in endogenous MUC2
PMA Up-regulates MUC2 Transcription via Ras, ERK, and NF-?B
by guest on December 26, 2015
Marvin H. Sleisenger and Young S. Kim
2002, 277:32624-32631. J. Biol. Chem.
Lee,Szymkowski, Sang-Young Han, Bong H.
Carol B. Basbaum, Nancy Q. Fan, David E.
Crawley, Jian-Dong Li, James R. Gum, Jr.,
Hae-Wan Lee, Dae-Ho Ahn, Suzanne C.
Intestinal Mucin via Ras, ERK, and NF-
Up-regulates the Transcription of
Phorbol 12-Myristate 13-Acetate
GENES: STRUCTURE AND
doi: 10.1074/jbc.M200353200 originally published online June 20, 2002
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