In this study, we demonstrate that forskolin⁄ 3-isobutyl-1-methylxanthine IBMX and the second messenger cAMP upregulate Cdx-2 promoter and endogenous Cdx-2 gene expression, specifically i
Trang 1expression of caudal homeobox gene Cdx-2 by cyclic AMP Liang Chen1,2, Peixiang Wang1,2, Cristiano F Andrade1,2, Ian Y Zhao1,2, Philip E Dube´3,
Patricia L Brubaker3,4, Mingyao Liu1,2,3and Tianru Jin1,2,4,5
1 Division of Cell and Molecular Biology, Toronto General Research Institute, University Health Network
2 Institute of Medical Science, University of Toronto, Canada
3 Department of Physiology, University of Toronto, Canada
4 Department of Medicine, University of Toronto, Canada
5 Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada
Homeodomain (HD) proteins, encoded by homeobox
genes, are tissue or cell-type specific transcription
fac-tors They are involved in embryogenesis; cell growth,
differentiation and apoptosis; hormone synthesis; and
many other biological and physiological cellular
events In the adult, the caudal HD protein Cdx-2 is
expressed in differentiated intestinal epithelia, inclu-ding the proglucagon producing endocrine L cells [1–5]
It is also expressed in the pancreatic islets, pancreatic insulin producing endocrine B cell lines, and proglu-cagon producing endocrine A cell lines [4–7] Using
‘knock out’ approaches, two research laboratories have
Keywords
Cdx-2; cAMP; Epac; ERK1 ⁄ 2; proglucagon
Correspondence
T Jin, Division of Cell and Molecular
Biology, Toronto General Research Institute,
University Health Network 67 College St.,
Toronto, Ontario, M5G 2M1
Fax: +1 416 340 3453
Tel: +1 416 340 4800, ext 4768
E-mail: tianru.jin@utoronto.ca
(Received 4 March 2005, accepted
31 March 2005)
doi:10.1111/j.1742-4658.2005.04694.x
Cdx-2 is a transactivator for the proglucagon gene in pancreatic and intes-tinal endocrine cells Cdx-2 is also expressed in differentiated intesintes-tinal epi-thelia of nonendocrine origin Cdx-2–⁄ – mice are embryonic lethal, while Cdx-2+⁄ – mutants show multiple malfunctions including the formation of intestinal polyps Within the polyps, the remaining wild type Cdx-2 allele ceases its expression, while the expression of both Cdx-2 and proglucagon
in the endocrine cells remains unaltered, indicating that Cdx-2 could be haplo-insufficient for nonendocrine cells, but not for proglucagon pro-ducing endocrine cells We propose that mechanisms underlying Cdx-2 expression and auto-regulation [Xu F, Li H & Jin T (1999), J Biol Chem
274, 34310–34316] differ in these two types of cells We show here that forskolin and cAMP upregulate Cdx-2 expression in proglucagon produ-cing cells, but not in colon cancer cells and primary intestinal cell cultures
It is unlikely that the activation is mainly mediated by PKA, because the activation was observed in a PKA deficient cell line Cotransfecting a dominant negative Ras expression plasmid substantially repressed the Cdx-2 promoter, in contrast to a previous finding that Ras is a negative factor for Cdx-2 expression in colon cancer cells Furthermore, forskolin activated ERK1⁄ 2 phosphorylation in the endocrine cells, and attenuation
of ERK1⁄ 2 phosphorylation by its inhibitor is associated with attenuated Cdx-2 expression Finally, an Epac pathway specific cAMP analogue stimulated both ERK1⁄ 2 phosphorylation and Cdx-2 expression Taken together, our observations suggest that Cdx-2 expression is regulated by the second messenger cAMP, cell-type specifically, via the Epac pathway
Abbreviations
CRE, cAMP response element; Epac, Exchange protein directly activated by cyclic AMP; FRIC, fetal rat intestinal cell; HD, homeodomain; IBMX, 3-isobutyl-1-methylxanthine; LUC, luciferase; MAPK, mitogen activated protein kinase; PKA, protein Kinase A; PKAc, catalytic subunit
of PKA.
Trang 2independently demonstrated that Cdx-2–⁄ – mice die
during 3.5–5.5 day post coitus (dpc) [8,9] Interestingly,
deleting one wild type Cdx-2 allele leads to multiple
malfunctions, including the development of polyp-like
lesions in the proximal colon [8,9] Consistently,
extensive in vitro and ex vivo studies by a number
of laboratories have identified more than two-dozen
potential downstream target genes of Cdx-2 [3–7,
10–25], including the genes that encode proglucagon
and insulin in pancreatic islets and intestinal endocrine
cells [4–6]
Within the intestinal polyps in the Cdx-2+⁄ – mice,
the remaining wild type Cdx-2 allele ceases its
expres-sion, while its expression in the surrounding normal
intestinal epithelia continues [8,9] However, the
expression of both Cdx-2 and proglucagon in the
endocrine cells of both pancreatic and intestinal origin
appears to be unaltered in the Cdx-2+⁄ – mice We
hypothesized that Cdx-2 expression could be
haplo-insufficient in selected types of cells [20]
To understand molecular mechanisms underlying this
intriguing cell-type specific event, we have isolated
the mouse Cdx-2 gene promoter, and initiated an
exami-nation of transcription factors and signaling
mole-cules that regulate the expression of this promoter We
found that cotransfection of the Cdx-2 cDNA led to
upregulation of the expression of the Cdx-2 gene
promo-ter in cell lines that express endogenous Cdx-2 [20] The
activation, however, was not observed when the naive
fibroblast cell lines were utilized [20] We also
demon-strated that the POU HD protein Oct-1 is able to bind
to the Cdx-2 gene promoter and is implicated in
regula-ting Cdx-2 promoter expression and auto-expression
[21] Such an auto-regulatory mechanism would provide
an explanation as to why one functional Cdx-2 allele is
sufficient for maintaining its own expression and for
regulating proglucagon gene expression in the
pancre-atic A and intestinal L endocrine cells It, however,
raises an even more intriguing question: why could one
functional Cdx-2 allele be insufficient in maintaining its
own expression in the intestinal nonendocrine cells, and
in preventing the formation of intestinal polyps for the
nonendocrine intestinal epithelia?
We propose that molecular mechanisms underlying
Cdx-2 expression, including its auto-expression, differ
in the endocrine cells from those in the nonendocrine
intestinal epithelia In this study, we demonstrate
that forskolin⁄ 3-isobutyl-1-methylxanthine (IBMX)
and the second messenger cAMP upregulate Cdx-2
promoter and endogenous Cdx-2 gene expression,
specifically in the proglucagon producing endocrine
cells It is unlikely that the activation is mainly
mediated by the protein kinase A (PKA) signaling
pathway In contrast, forskolin⁄ IBMX does not acti-vate Cdx-2 promoter and⁄ or endogenous Cdx-2 gene expression in nonendocrine colon cancer cell lines and in primary fetal rat nonendocrine intestinal cell cultures Our results also suggest that the exchange protein directly activated by cAMP (Epac) signaling pathway is involved in this cell type specific activa-tion event
Results
Forskolin/IBMX upregulates Cdx-2 gene promoter
in proglucagon producing endocrine cell lines
We started our investigation by seeking for chemicals
or signaling molecules that upregulate Cdx-2 promoter expression in the proglucagon producing cell lines only The )769 Cdx-2 ⁄ LUC fusion gene plasmid [20,21] was transfected into the proglucagon producing GLUTag cell line After the transfection, RA (1 lm), TPA (1 lm), or forskolin⁄ IBMX (F ⁄ I, 10 lm each) was added to the medium The cells continued to grow for 20 h before harvested for LUC reporter gene analysis As can be seen in Fig 1A, RA and TPA had virtually no effect on Cdx-2 promoter expression (panel i), while forskolin⁄ IBMX treatment caused approximately 2.5-fold activation (panel ii) Similar results were obtained for two other endocrine cell lines, InR1-G9 and STC-1 (see below, and data not shown)
On the other hand, when the same )769 Cdx-2 ⁄ LUC fusion reporter construct was transfected into the three nonendocrine colon cancer cell lines, we did not observe any appreciable activation by forskolin⁄ IBMX treatment A representative result on the HT-29 cell line is shown in Fig 1A, panel iii Thus, it seems that forskolin⁄ IBMX has different effects on Cdx-2 promo-ter expression between endocrine cells and nonendo-crine cells We then further examined the effect of forskolin⁄ IBMX treatment for different time lengths
on Cdx-2 promoter expression in the GLUTag cell line It was found that the activation appeared at 4 h and gradually increased during the 20 h experimental period (Fig 1A, panel iv) Panel v shows that forsko-lin⁄ IBMX generated no substantial effect on the expression of pBLUC, the promoter-less plasmid util-ized in the construction of both Cdx-2⁄ LUC and GLU⁄ LUC [4,20]
Forskolin/ IBMX activates endogenous Cdx-2 mRNA expression in endocrine cell lines
We then asked the question whether forskolin⁄ IBMX treatment would stimulate endogenous Cdx-2 mRNA
Trang 3expression After the endocrine cell lines were treated
for the indicated period of time, total cell RNA was
extracted and analyzed by northern blotting We found
that, consistent with the data from our LUC reporter
gene assay, Cdx-2 mRNA expression was notably
activated in the GLUTag cell line by 8–24 h treatment with forskolin⁄ IBMX (Fig 1B)
A previous study has shown that the expression of proglucagon mRNA in the InR1-G9 cell line cannot
be activated by forskolin⁄ IBMX [26], indicating that
i) GLUTag
A
2
**
*
1
0
3
2
1
0 0
0.4 0.8 1.2
1
0
Reporter
BLUC
-769 Cdx-LUC
20
TPA RA
Time (h)
2
1
0
B
GLUTag
C C 8 8 24 24 Time (h)
Cdx-2
Tub
1 3.1 2.9
C
Time (h)
Cdx-2
Tub
1 1.4 2.7 2.6 3.7 4.3
InR1-G9
C C 2 2 4 4 6 6 12 12 24 24
D
Time (h) Cdx-2
Tub
1 0.9 0.9 1.0 0.9 1.0
Caco-2
C C 2 2 4 4 6 6 12 12 24 24
Fig 1 Forskolin ⁄ IBMX activates Cdx-2 promoter and Cdx-2 mRNA expression in the GLUTag cell line (A) Forskolin ⁄ IBMX (panels ii, iv), but not TPA or RA (panel i), activated Cdx-2 promoter expression in GLUTag cells The activation was not observed for the colon cancer cell line HT-29 (panel iii), nor for the promoter-less control LUC reporter pBLUC (panel v) Indicated cell lines were transfected with 3 lg )769 Cdx-2 ⁄ LUC fusion gene plasmid ( )769 Cdx-LUC), or pBLUC All trans retinoic acid (RA, 1 l M ), TPA (1 l M ), or forskolin ⁄ IBMX (F ⁄ I, 10 l M each), or ethanol (vehicle, V) was added 20 h before the cells were harvested for LUC reporter gene analysis Relative LUC activity was calculated as the fold increase with the activity in the vehicle treated cells defined as onefold (mean ± SE, n ¼ 3) (B–D) GLUTag (B), InR1-G9 (C), and Caco-2 (D) cell lines were treated with either control medium (with ethanol as the vehicle, V), or medium with 10 l M forskolin plus 10 l M IMBX (F ⁄ I) at indica-ted h before harvesting Total RNA was extracindica-ted for northern blot analysis using cDNA probes for hamster Cdx-2 (Cdx-2) or mouse tubulin (Tub) as the loading control F ⁄ I, forskolin ⁄ IBMX.
Trang 4this cell line is PKA deficient For this cell line, we
therefore included additional time points to examine
the effects of forskolin⁄ IBMX As shown in Fig 1C,
2 h after forskolin⁄ IBMX treatment, Cdx-2 mRNA
expression started to arise Substantial activation was
observed 4 h after the treatment, and the elevated
Cdx-2 mRNA expression was maintained during the
whole 24 h experimental procedure Considering the
PKA deficient nature of this cell line, the above
obser-vation would suggest that the actiobser-vation by
forsko-lin⁄ IBMX on Cdx-2 expression in the proglucagon
producing endocrine cell lines is not a PKA dependent
event (see further examination below)
In an effort to examine Cdx-2 expression profiles in
different cell lines, we were unable to detect Cdx-2
mRNA expression by northern blotting in two colon
cancer cell lines, HT-29 and SW480 (data not shown)
We, however, could detect Cdx-2 mRNA expression in the human colon cancer Caco-2 cell line As shown in Fig 1D, forskolin⁄ IBMX treatment generated no sub-stantial effect on Cdx-2 mRNA expression in the Caco-2 cell line
We then developed a real time RT-PCR approach
to examine the effect of forskolin⁄ IBMX on Cdx-2 mRNA expression in the HT-29 cell line, while the gut endocrine GLUTag cell line was utilized as the positive control As shown in Fig 2A, in HT-29 cells there is
no significant effect on Cdx-2 mRNA expression by forskolin⁄ IBMX treatment for 2, 4, 6, and 16 h In contrast, Cdx-2 mRNA expression in the GLUTag cell line was significantly activated by an 8- h treatment (Fig 2B), indicating that the real time RT-PCR
HT-29/Cdx-2
GLUTag/Cdx-2 15
10
**
*
*
*
5
0 1
2 3 4
0
6 5 4 3 2 1 0
Treatment
0 2
4
Fig 2 Examination of the effect of
forskolin ⁄ IBMX on Cdx-2 mRNA expression
in HT-29 cell line by real time RT-PCR The
nonendocrine colon cancer cell line HT-29
(A), or the endocrine cell line GLUTag (B), or
the primary cell culture FRIC (C,D) were
treated with either the control medium (with
ethanol as the vehicle, V), or the same
med-ium plus 10 l M forskolin and 10 l M IBMX
(F ⁄ I) at indicated h before harvesting Total
RNA was extracted and real time RT-PCR
experiments were conducted against Cdx-2
genes (A–C) or proglucagon gene (D), as
described in the Experimental procedures
section Results obtained were
normal-ized using GAPDH as the control (Cdx-2⁄
GAPDH) N ¼ 4 *P < 0.01; **P < 0.005.
Trang 5approach utilized here could detect elevated Cdx-2
mRNA expression
The nonendocrine cell lines utilized in this study are
colon cancer cells of human origin To investigate
whe-ther the lack of response to forskolin⁄ IBMX on Cdx-2
expression in these cell lines is associated with their
cancerous status, we conducted further examination
using the primary fetal rat intestinal cell (FRIC)
cul-tures It has been reported that 99% of FRIC cells
represent the nonendocrine intestinal cells, while 1%
population represents the proglucagon producing
endocrine L cells [27] Proglucagon mRNA expression
in the endocrine L cell lines and in the FRIC cultures
can be activated by forskolin⁄ IBMX [28–31] As
shown in Fig 2C, Cdx-2 mRNA expression in the
FRIC cultures was not apparently affected by
forsko-lin⁄ IBMX treatment for 2 and 6 h, and the 24-h
treat-ment repressed Cdx-2 mRNA expression On the other
hand, proglucagon mRNA expression in the FRIC
cul-tures was significantly stimulated by forskolin⁄ IBMX
treatment for 6 and 24 h (Fig 2D) The above real
time RT-PCR results further supported our suggestion
that forskolin⁄ IBMX specifically upregulates Cdx-2
expression in the proglucagon producing endocrine
cells Forskolin⁄ IBMX may repress, or at least not
activate, Cdx-2 expression in nonendocrine intestinal
cells
It should be pointed out that when FRIC cells were
examined using the real time RT-PCR approach, large
S.D values were generated in each set of assay This
is understandable because of the heterogeneity of the
FRIC cultures When, however, HT-29 was examined,
the S.D values in each set of experiments were also
relatively high We have no proper explanation for this
observation at this time
Forskolin⁄ IBMX or cAMP stimulates Cdx-2
protein expression in the proglucagon producing
endocrine cells
We next examined whether forskolin⁄ IBMX activates
Cdx-2 protein expression The GLUTag cell line was
examined first As shown in Fig 3A, after a 3 h
treat-ment, forskolin⁄ IBMX substantially activated Cdx-2
protein expression To our surprise, virtually no
activa-tion was observed after 6 or 12 h treatment (Fig 3A)
This is in contrast with the activation profile by
forsk-olin⁄ IBMX treatment at the Cdx-2 mRNA level
(Fig 1B,C) We then focused on assessing the
activa-tion within 4 h period Figure 3B shows the substantial
activation by forskolin⁄ IBMX treatment on Cdx-2
protein expression in the GLUTag cell line at 1, 2, 3,
and 4 h
To investigate whether the activation of Cdx-2 pro-tein expression by forskolin⁄ IBMX is caused by increasing intracellular levels of cAMP, the cell mem-brane permeable cAMP analogue, 8-Br-cAMP, was utilized for the InR1-G9 cell line (Fig 3C) After the cells were treated with forskolin⁄ IBMX or 8-Br-cAMP for 2 or 4 h, Cdx-2 protein expression was elevated substantially, suggesting that the intracellular level of cAMP plays a role in the regulation of Cdx-2 expres-sion To our surprise, the cell membrane permeable cGMP analogue, 8-Br-cGMP, considered as a negative control in our experimental design, also activated Cdx-2 protein expression substantially (Fig 3C) The above observations also indicated that the vehicle utilized in this study (from a 1 to 12 h period) had virtu-ally no effect on Cdx-2 protein expression Our further examinations were then conducted using the vehicle as the control for the longest time point Figure 3D shows the activation of forskolin⁄ IBMX treatment on Cdx-2 protein expression in the gut endocrine STC-1 cell line
at 1, 2 and 4 h, but not at 6 h We then examined the effect of forskolin⁄ IBMX on Cdx-2 protein expression
in the Caco-2 cell line As shown in Fig 3E, no activa-tion was observed In conducting this examinaactiva-tion, we included the Epac pathway specific cAMP analogue 8-pMeOPT-2¢O-Me-cAMP This analogue also generated
no stimulatory effect on both Cdx-2 protein expression and ERK1⁄ 2 phosphorylation (see below)
The activation may not be mediated by PKA
As indicated above, the InR1-G9 cell line may carry a defect in its PKA signaling pathway Forskolin⁄ IBMX failed to stimulate the expression of proglucagon gene mRNA and its promoter in this cell line, in contrast
to significant activation by forskolin⁄ IBMX in primary pancreatic islet cell cultures or in intestinal endocrine
L cell lines [26,29–31] Based on those observations, we further investigated the involvement of PKA in Cdx-2 expression First, we compared the effects of PKAc cotransfection on the expression of the Cdx-2 promo-ter vs the proglucagon gene promopromo-ter PKAc cotrans-fection generated no significant effect on Cdx-2 promoter expression in InR1-G9 (Fig 4A, left panel) and GLUTag cells (data not shown) PKAc cotransfec-tion, however, significantly activated the expression of the proglucagon gene promoter in InR1-G9 (Fig 4A, right panel) and GLUTag cells (data not shown) Similar to what has been observed for the GLUTag cell line shown in Fig 1A, forskolin⁄ IBMX treatment activated Cdx-2 gene promoter, approximately 2.5-fold, in the InR1-G9 cell line (Fig 4B) Further LUC reporter gene analyses were conducted using different
Trang 6sized Cdx-2⁄ LUC fusion gene constructs [20] for both
InR1-G9 and GLUTag cell lines for the identification
of cis-element(s) that mediates forskolin⁄ IBMX
treat-ment, and our results indicate that it is unlikely that a putative CRE element in the Cdx-2 gene promoter is involved (data not shown)
Time (h)
Time (h) Cdx-2
Cdx-2
Actin Actin
Actin F/I cGMP cAMP V fold
ERK pERK Actin Cdx-2
fold Actin
- -
- -
+ +
+ +
+ + + +
+ +
+ +
1 92 3.1 2.9 1.1
V 1 2 4 6
1
1
1 2 4 1 2 4 1 2 4
1.9 2.9 2.7 2.4 2.3 2.7
3.9 2.7 1.4 0.7
1 94 99 fold
fold
V
V
F/I
F/I
1.2 1.2
1 3.6 1 2.9 1 3.7 1 3.5
Time (h)
1 1 2 2 3 3 4 4 3
A
C
E
B
D
6
Caco-2
Fig 3 Comparison of the effect of forkolin ⁄ IBMX on Cdx-2 protein expression in endocrine cell lines vs the nonendocrine HT-29 cell line (A,B) GLUTag cells were grown in the presence of ethanol (vehicle, V) or 10 l M forskolin plus 10 l M IBMX (F ⁄ I) for the indicated h before the cells were harvested for examination of Cdx-2 protein expression by western blotting The same membranes were stripped and followed
by hybridization with an anti-(b-actin) Ig (loading control) (C) InR1-G9 cells were grown in the presence of ethanol (vehicle, V) or 10 l M forsk-olin plus 10 l M IBMX (F ⁄ I), or 0.25 m M 8-Br-cGMP (cGMP), or 0.25 m M 8-Br-cAMP (cGMP), for the indicated h before the cells were harves-ted for examination of Cdx-2 protein expression by western blotting The same membranes were stripped and hybridized with an anti-(b-actin) Ig (D) The response to forskolin ⁄ IBMX treatment on Cdx-2 expression in the STC-1 cell line (E) Caco-2 cells were treated with 10 l M forskolin and IBMX, or 10 l M Epac pathway specific cAMP analogue (8-pMeOPT-2¢O-Me-cAMP) and their effects on Cdx-2 expression and ERK1 ⁄ 2 phosphorylation were examined Antibodies against b-actin and total ERK1 ⁄ 2 were utilized to ensure equal loading.
Trang 7We then investigated whether the InR1-G9 cell line
is indeed PKA deficient, and whether PKAc
cotrans-fection would trigger the PKA pathway in this cell
line We examined the PKA kinase activity in PKAc
transfected InR1-G9 cells along with untransfected InR1-G9 and GLUTag cells A representative result is shown in Fig 4C Without the PKAc transfection, PKA kinase activity in the InR1-G9 cell line was barely detectable, compared with that detected in the GLUTag cell line Nevertheless, the PKA protein expression for both cell lines was readily detected by Western blotting (data not shown) However, when the InR1-G9 cells were transfected with the PKAc expres-sion plasmid, the PKA kinase activity was significantly elevated These results confirm that the InR1-G9 cell line is defective in PKA activity and cotransfection of PKAc restores its PKA activity
Finally we tested the effect of H-89, a known inhib-itor of PKA, on the expression of both the progluca-gon gene promoter and the Cdx-2 gene promoter in the PKA active GLUTag cell line As shown in Fig 5, the effects of H-89 on these two promoters are appar-ently different At the concentrations of either 0.1 or
1 lm, H-89 significantly repressed the basal expression
of the proglucagon gene promoter, although forskolin⁄ IBMX stimulated expression cannot be completely blocked (left panel) However, at the same concentra-tions, H-89 failed to repress the Cdx-2 promoter Thus, H-89 inhibits the proglucagon gene promoter presuma-bly by blocking the PKA signaling pathway That the expression of the Cdx-2 gene promoter was not affected
by H-89, is probably due to the fact that pathway(s) other than PKA is⁄ are responsible for mediating the activation by forskolin⁄ IBMX and membrane per-meable cAMP treatment
Dominant negative Ras represses the Cdx-2 gene promoter
Numerous recent reports suggested that the second messenger cAMP may use the cAMP-Epac-Ras⁄ Rap-MEK-MAPK signaling pathway to regulate gene expression and other cellular functions [32,33] In addition, it has been suggested that in colon cancer cell lines, Cdx-2 expression could be down regulated
by oncogenic Ras [34] We therefore investigated the effect of a dominant negative Ras molecule on
Cdx-2 promoter expression in the proglucagon producing endocrine cell lines Figure 6 shows that the domin-ant negative Ras significdomin-antly inhibited Cdx-2 pro-moter in the InR1-G9 cell line (right panel) In contrast, significant repression was not observed when either the TK promoter (data not shown) or the proglucagon gene promoter (left panel) was tes-ted These results indicate that Ras could be a posit-ive factor for Cdx-2 expression in proglucagon producing endocrine cells
InR1-G9
InR1-G9
200
100
0 Rep
PKAc
769 Cdx-LUC
2
1
0
InR1-G9
NS
NS
**
**
*
A
InR1-G9 2
6
4
2
0
1
0
Relative LUC Activity Relative LUC Activity
Rep
pCDNA3 pCDNA3
0
0 5 -769 Cdx-LUC -472 GLU-LUC
.5
1.0 1.0
0
0
.5 5
1.0 1.0
Rep
Fig 4 PKAc cDNA cotransfection has different effects on Cdx-2
promoter vs proglucagon gene promoter expression in the PKA
deficient InR1-G9 cell line (A) InR1-G9 cells were cotransfected
with 3.0 lg )769 Cdx-LUC (left panel) or 3.0 lg )472 GLU-LUC
(right panel), plus the indicated amount of PKAc and ⁄ or pCDNA3
(vector for PKAc) Cells were harvested 20 h after the transfection,
and relative LUC reporter gene activity was calculated as the fold
increase with the activity in the cells received no PKAc
transfec-tion, defined as onefold (mean ± SE, n ¼ 3) (B) InR1-G9 cells were
transfected with 3 lg )769 Cdx-2 ⁄ LUC fusion gene plasmid ()769
Cdx-LUC) Forskolin ⁄ IBMX (F ⁄ I, 10 l M each), or ethanol (vehicle, V)
was added 20 h before the cells were harvested for LUC reporter
gene analysis Relative LUC activity was calculated as the fold
increase with the activity in the vehicle treated cells defined as
onefold (mean ± SE, n ¼ 3) (C) PKA activities in GLUTag and
InR1-G9 cells, and PKAc transfected InR1-InR1-G9 cells were assayed.
Approximately 3.0 mg total cell lysates were utilized for
immuno-precipitation, and one third of the precipitate was used for the PKA
assay Relative PKA activity in untransfected and PKAc transfected
InR1-G9 cells was calculated as the percentage of that obtained
from GLUTag cells (mean ± SE, n ¼ 5).
Trang 8Forskolin/IBMX stimulates ERK1/2
phosphoryla-tion in proglucagon producing cell lines
As a small GTPase, Ras may utilize MAP kinase to
carry out its signaling and biological functions We
then investigated the effect of forskolin⁄ IBMX
treat-ment on the phosphorylation status of one of the
MAP kinases, ERK1⁄ 2 in proglucagon producing cell
lines
Forskolin⁄ IBMX treatment was found to stimulate ERK1⁄ 2 phosphorylation in GLUTag, STC-1, and InR1-G9 cell lines A representative result from the InR1-G9 cell line is shown in Fig 7A The expression level of phosphorylated ERK1⁄ 2 was elevated 5 min after forskolin⁄ IBMX treatment, while the effect on total ERK1⁄ 2 expression was not appreciable In this particular experiment, forskolin⁄ IMBX treatment for
120 min did not activate ERK1⁄ 2 phosphorylaton (lane 5) We, however, observed substantial activation for this time course for three other experiments (data not shown) The activation of ERK1⁄ 2 phosphoryla-tion by forskolin⁄ IBMX treatment was associated with elevated Cdx-2 protein expression, consistent with the result shown in Fig 3B When PD98059, a MEK-1 inhibitor, was included, forskolin⁄ IBMX mediated ERK1⁄ 2 phosphorylation was significantly inhibited, starting from 30 min (lanes 9–12) PD98059 also inhib-ited the basal expression of phosphorylated ERK1⁄ 2 (comparing lane 1 with lane 7)
Although the basal expression of phosphorylated ERK1⁄ 2 was inhibited by PD98059, the basal Cdx-2 expression was not affected (comparing lane 7 with lane 1) However, PD98059 inhibited activated Cdx-2 protein expression by forskolin⁄ IBMX treatment for
4 h (comparing lane 12 with lane 6), despite the absence of the repression at the other time courses It should be pointed out that if phosphorylated ERK1⁄ 2 mediates forskolin⁄ IBMX stimulated Cdx-2 transcrip-tion, further phosphorylation events on transcription factor(s) should be involved Inhibition of ERK1⁄ 2 phosphorylation by PD98059, may not affect the fac-tors⁄ mediators that have already been phosphorylated
4
3
2
1
0
4
3
2
1
0
Glu/LUC
1 1 0.1 0.1
1 1 0.1 0.1 Cdx-2/LUC
Fig 5 Comparison of the effect of H-89 on
expression of the proglucagon and Cdx-2
gene promoters Three micrograms )476
GLU-LUC (left panel) or )769 Cdx-2-LUC
reporter gene plasmid was transfected into
the GLUTag cell line Ten micromolar
forsko-lin plus 10 l M IBMX (F ⁄ I) was added to the
cells 20 h before harvesting, with or without
indicated amount of H-89 (added 45 min
before the addition of forskolin ⁄ IBMX).
Relative LUC reporter gene activity was
calculated as the fold increase with the
activity in the untreated cells defined as
onefold (mean ± SE, n ¼ 3).
1.2
1.8
1.4
-+
+ +
+
+
0.5
1.0
1.5 NS
**
Fig 6 The effect of a dominant negative ras cDNA cotransfection
on Cdx-2 promoter expression InR1-G9 cells were cotransfected
with 3 lg )476 GLU ⁄ LUC (left panel) or )769 Cdx-2 ⁄ LUC, and
1.5 lg pCDNA3 (vector), or 1.5 lg dominant negative ras cDNA.
Cells were harvested 20 h later for LUC reporter gene analysis
Rel-ative LUC reporter gene activity was calculated as the fold increase
with the activity in the untreated cells defined as onefold (mean ±
SE, n ¼ 3).
Trang 9by active ERK1⁄ 2 Therefore, it is reasonable to accept
a 2- to 3-h time delay to attenuate Cdx-2 protein
expression in response to the inhibition on ERK1⁄ 2
phosphorylation (see the Discussion section for our
further interpretation)
Epac pathway specific cAMP analogue stimulates
Cdx-2 protein expression and ERK 1/2
phosphorylation
Finally, we initiated an examination of whether the
Epac signaling pathway [35,36] specific cAMP
ana-logue would also stimulate Cdx-2 protein expression
in the InR1-G9 cell line As shown in Fig 7B,
10 lm 8-pMeOPT-2¢O-Me-cAMP generated comparable
effects on both Cdx-2 protein expression and ERK1⁄ 2
phosphorylation to that induced by forskolin⁄ IBMX treatment As we have already presented in Fig 3E, such stimulatory effects were not observed when the colon cancer cell line Caco-2 was examined
Discussion
Several groups have investigated signaling molecules or pathways that may up or down regulate Cdx-2 expres-sion in the nonendocrine intestinal epithelia [34,37–41] Cdx-2 expression was found to be down regulated by the oncogenic Ras [34], and up regulated by butyrate [39] It has also been reported that Cdx-2 expression cannot be detected in the adnomatous polyposis coli (APC) mutated colon cancer cell lines, while introducing wild type APC cDNA into an APC mutated cell line rendered it to re-express Cdx-2 mRNA [37] However,
a recent immunohistochemistry study indicated that Cdx-2 expression in the mouse gut was not altered by APC or Ras status, or by butyrate treatment [40] Another study by Kim et al linked Cdx-2 expression with the tumor suppressor PTEN (phosphatase and tension homologue deleted from chromosome 10), and the phosphatidylinositol 3-kinase (PI3K) signaling path-way [38] However, little is known about signaling mole-cules and pathways that may regulate Cdx-2 expression
in the proglucagon producing endocrine cells
In this study, we examined the effects of forsko-lin⁄ IBMX on Cdx-2 expression, and investigated pos-sible signaling pathways that mediate such expression
We found that Cdx-2 expression could be activated by forskolin⁄ IBMX Using a reporter gene assay and nor-thern blotting, we demonstrated that this activation occurs at the transcriptional⁄ mRNA level Addition-ally, Cdx-2 protein expression was also elevated by forskolin⁄ IBMX as detected by western blotting However, the activation only takes place in the pro-glucagon producing endocrine cell lines, but not in the nonendocrine colon cancer cell lines Those data collec-tively suggested that activation of Cdx-2 expression by forskolin⁄ IBMX occurs in a cell type specific manner The InR1-G9 cell line may be PKA deficient [26]
We found that indeed PKA kinase activity in this cell line is significantly low, compared with that in the GLUTag cell line (Fig 4C) The observations that forskolin⁄ IBMX activates the Cdx-2 promoter with or without a putative CRE element (data not shown), and that Cdx-2 promoter and endogenous Cdx-2 mRNA and protein expression in the InR1-G9 cell line were also activated by forskolin⁄ IBMX treatment (Figs 1C, 3C and 4B), led to the hypothesis that the activation is not mediated by PKA We found that in three proglucagon producing cell lines, cotransfection
InR1-G9
A
B
F/I (min)
PD98059
InR1-G9
V F/I Epac
Cdx-2 Actin
pERK ERK
Cdx-2 Actin pERK1/2 ERK1/2
1 2 3 4 5 6 7 8 9 10 11 12
-1 2 4 -1 2 4 1 2 4
- - - -
-+ + + + + +
5 30 60 120 240 5 30 60 120 240
Fig 7 The effect of forskolin ⁄ IBMX and Epac pathway specific
cAMP analogue on the phosphorylation status of ERK1 ⁄ 2 and
Cdx-2 protein expression A InR1-G9 cells were grown in the presence
of ethanol (vehicle for forskolin ⁄ IBMX, V) or 10 l M forskolin plus
10 l M IBMX (F ⁄ I) for the indicated time before the cells were
harvested for examination of Cdx-2 and phosphorylated ERK ⁄ 1 ⁄ 2
expression by western blotting For one set of cells, the MEK
inhib-itor PD98059 (50 l M ) was added 45 min before the addition of F ⁄ I.
The same membranes were stripped and followed by hybridization
with an anti-(b-actin) Ig (loading control), and the antibody against
total ERK1 ⁄ 2 B InR1-G9 cells were grown in the presence of
10 l M 8-pMeOPT-2¢O-Me-cAMP [F ⁄ I as the positive control, and
vehicle (V) as the negative control] Cdx-2 expression and ERK1 ⁄ 2
phosphorylation were then examined Antibodies against b-actin
and total ERK1 ⁄ 2 were utilized to ensure equal loading.
Trang 10of a dominant negative Ras expression plasmid
significantly repressed Cdx-2 expression, but not the
proglucagon gene promoter, indicating that Ras is a
positive factor This is in contrast with the previous
report that Ras is a negative factor for Cdx-2
expres-sion in colon cancer cell lines [34] In addition, we
found that forskolin⁄ IBMX specifically stimulates
ERK1⁄ 2 phosphorylation in proglucagon producing
cell lines, and MEK inhibition attenuated
forsko-lin⁄ IBMX activated Cdx-2 protein expression at 4 h
(Fig 7) These observations collectively suggest the
involvement of cAMP-Epac-Ras⁄ Rap-MEK-MAPK
signaling pathway (see below)
We noticed the discrepancy regarding the effects of
forskolin⁄ IBMX on Cdx-2 expression at the mRNA
and protein levels First, the activation of Cdx-2
pro-tein expression could be observed as early as 30 min
Such an early response would suggest that, in addition
to stimulating Cdx-2 mRNA expression, forskolin⁄
IBMX might also play a role in stabilizing Cdx-2
protein Consistently, we found that PD98059 repressed
forskolin⁄ IBMX activated Cdx-2 protein expression
only at 4 h, but not within the first 2 h One may
specu-late that forskolin⁄ IBMX may stabilize Cdx-2 protein
via a yet to be identified mechanism, and this effect
cannot be blocked (or immediately blocked) by MEK
inhibition Activated ERK1⁄ 2, however, may stimulate
Cdx-2 transcription via phosphorylating its
transcrip-tional activators, and this event would take longer time,
and it could be blocked by MEK inhibition Second,
activation by forskolin⁄ IBMX at the protein level was
not observed beyond 4 h However, the activation at
the mRNA level was detectable by northern blot
analy-sis during the whole 2–24 h experimental period for the
InR1-G9 cell line (Fig 1C) One may postulate the
existence of a negative feedback loop at the Cdx-2
pro-tein expression level to explain such a difference To
our surprise, both 8-Br-cAMP and 8-Br-cGMP were
found to activate Cdx-2 protein expression in InR1-G9
cells A possible explanation would be the involvement
of a nucleotide gated ion channel [32,42,43] in these
endocrine cell lines We made an attempt to identify
the existence of such channel in the InR1-G9 cell line
without success However, we did observe that treating
InR1-G9 cells with 45 mm potassium chloride
(indu-cing membrane depolarization) led to enhanced Cdx-2
protein expression The discrepancy between the
responses of Cdx-2 protein and mRNA expression to
forskolin⁄ IBMX treatment further indicated the
comp-lexity of the corresponding regulatory networks One
may suggest that this discrepancy and the presence of
negative feedback loops may partially explain why
opposite results have been obtained by different research
groups in assessing the role of APC, Ras status, and butyrate treatment on Cdx-2 expression [34,37–41]
An important observation in this study is that the activation of Cdx-2 promoter by forskolin⁄ IBMX took place only in the proglucagon producing endocrine cells Such a finding supported our overall hypothesis that signaling molecules and⁄ or pathways involved in Cdx-2 expression differ in the endocrine cells from that in the nonendocrine intestinal epithelia However, as the non-endocrine cell lines utilized in this study are cancerous cells of human origin, we employed the primary FRIC culture system for conducting real time RT-PCR exami-nations (Fig 2) A majority of epithelial cells (99%) in FRIC cultures are nonendocrine [28,29] Such cultures therefore would represent primary nonendocrine intes-tinal cells Lack of the response to forskolin⁄ IBMX treatment in the FRIC cultures on Cdx-2 mRNA expression supported our overall hypothesis that the second messenger cAMP activates Cdx-2 expression only in the proglucagon producing endocrine cells Although the second messenger cAMP has long been shown to mediate specific intracellular signaling events through PKA [44,45], more recent observations have suggested that PKA does not account for all of the intracellular targets of cAMP, especially in endocrine cells [32,33,42,43] Furthermore, the identification of novel cAMP binding proteins that exhibit guanine nucleotide exchange (GEF) activities (cAMP-GEFs, or Epac) has opened a new research direction for under-standing the function of the second messenger cAMP [32,35,36,40] A GEF molecule may serve as a bridge between cAMP and small GTPases, including Ras, Rap1 and Rap2, leading to the activation of Raf and the mitogen activated protein kinase (MAPK) signaling pathway [32] The observation that ras may serve as a positive factor for Cdx-2 expression (Fig 6), and both the Epac pathway specific cAMP analogue and forsko-lin⁄ IBMX stimulate ERK1 ⁄ 2 phosphorylation and Cdx-2 expression collectively suggest the involvement of the Epac-Ras⁄ Rap-ERK pathway in regulating Cdx-2 expression To examine the expression profile of Epac molecules in pancreatic and intestinal proglucagon pro-ducing cells, and to identify whether Ras and⁄ or Rap are indeed involved in regulating Cdx-2 expression would further our understanding on this cell type spe-cific gene expression event
Experimental procedures
Materials
Tissue culture medium, calf serum and oligonucleotides were purchased from Invitrogen Life Technology Inc