Goblet cell-specific expression mediated by the
MUC2 mucin gene promoter in the
intestine of transgenic mice
James R.
Gum Jr.1,2,
James W.
Hicks3,
Anne-Marie
Gillespie4,
Elaine J.
Carlson4,
Lazlo
Kömüves5,
Satyajit
Karnik1,
Joe C.
Hong3,
Charles J.
Epstein4, and
Young S.
Kim1,3
1 Gastrointestinal Research
Laboratory, Department of Veterans Affairs Medical Center, San
Francisco 94121; and Departments of
2 Anatomy,
3 Medicine,
4 Pediatrics, and
5 Dermatology, University of
California at San Francisco, California 94143
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ABSTRACT |
The regulation of
MUC2, a major goblet cell mucin gene,
was examined by constructing transgenic mice containing bases
2864 to +17 of the human MUC2
5'-flanking region fused into the 5'-untranslated region of
a human growth hormone (hGH) reporter gene. Four of eight transgenic
lines expressed reporter. hGH message expression was highest in the
distal small intestine, with only one line expressing comparable levels
in the colon. This contrasts with endogenous
MUC2 expression, which is expressed at
its highest levels in the colon. Immunohistochemical analysis indicated
that goblet cell-specific expression of reporter begins deep in the crypts, as does endogenous MUC2 gene
expression. These results indicate that the
MUC2 5'-flanking sequence
contains elements sufficient for the appropriate expression of
MUC2 in small intestinal goblet cells.
Conversely, elements located outside this region appear necessary for
efficient colonic expression, implying that the two tissues utilize
different regulatory elements. Thus many, but not all, of the elements
necessary for MUC2 gene regulation reside between bases
2864 and +17 of the 5'-flanking region.
differentiation; gene expression; granular goblet cells; colon; human growth hormone reporter
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INTRODUCTION |
THE MAMMALIAN INTESTINAL mucosa undergoes constant and
rapid renewal throughout life and provides an excellent opportunity for
the study of gene expression and differentiation in highly specialized
cells (8). Small intestinal epithelial cells begin their existence as
the daughter cells of one or a small number of stem cells located
approximately four cell positions from the base of the crypts. These
cells differentiate into enterocytes, Paneth cells, enteroendocrine
cells, and goblet cells. Paneth cells migrate to the base of the
crypts, whereas the other cell types travel upward, passing through the
crypt-villus junction and ultimately transversing the entire length of
the villus before undergoing a degenerative process and being sloughed
into the lumen. The continuum of differentiation has been best studied in enterocytes, the most abundant cell type in the intestinal epithelium. Most enterocyte-specific gene products are first expressed as the migrating cells approach the crypt-villus junction,
approximately concurrently with the cessation of cell division (for
reviews, see Refs. 11 and 36). Goblet cells and enteroendocrine cells, however, are visible at lower positions in the crypts, indicating an
earlier onset of differentiation. Paneth cell differentiation also
occurs as the cells migrate, with the most mature cells being located
near the apex of the base of the crypts. Paneth cells have considerably
longer lifetimes than do the other cells of the intestinal epithelium,
suggesting unique patterns of growth and differentiation for this
lineage (4, 6, 32).
Much attention has been focused on identifying the factors and
sequences that regulate the transcription of enterocyte-specific genes.
Studies performed examining expression of promoter-reporter chimeras in
transgenic mice have been particularly informational as only in vivo
can the exquisite patterns of differentiation found in the intestinal
epithelium be completely achieved. Two fatty acid binding protein genes
designated liver fatty acid binding protein
(Fabpl) and intestinal fatty acid
binding protein (Fabpi), as well as
the sucrase-isomaltase gene (Si),
have been especially well studied using transgenic systems (9, 28, 29,
34, 38). These studies have enabled the identification of regulatory sequences important for establishing and maintaining proper gradients of gene expression along the horizontal (cephalocaudal) and vertical (crypt-villus) axes of the gut for each of these genes. In addition, sequences important for suppressing inappropriate expression have been
identified, as have nuclear proteins that function in regulation. These
studies have not only revealed important mechanisms for the regulation
of these particular genes but have also shed considerable insight on
the characteristics of gut stem cells, lineage allocation, and the
factors that regulate differentiation.
The present work focuses on the regulation of a goblet cell-specific
gene, MUC2.
MUC2 encodes a large (>5,000 amino
acid residue) gel-forming mucin abundantly expressed by intestinal
goblet cells and certain other mucus-secreting cells in normal,
diseased, and neoplastic tissues (12, 16, 17, 35). The
5'-flanking region of the MUC2
gene has been isolated as well, and has been shown to have promoter
activity in cultured cells (13, 26, 39). It is difficult to study the
factors responsible for the regulation of this gene in cultured cells,
however, in part because transfectable cell lines expressing the high
levels of MUC2 found in intestinal goblet cells are nonexistent. To circumvent this problem, we have examined the expression of a
MUC2-human growth hormone (hGH)
reporter-promoter construct in multiple pedigrees of transgenic mice.
This has allowed for examination of
MUC2 expression in fully
differentiated cell types.
The results of this study indicate that elements important for the
goblet cell-specific expression of
MUC2 reside in the 5'-flanking region of the gene. Examination of the pattern of reporter expression has revealed, however, important differences from that of the native
MUC2 gene. Moreover, the levels of
reporter expression exhibited by the different pedigrees varied
markedly, indicating significant integration site dependence and
lending credence to the notion that there is regulatory significance to
the clustering of secretory mucin genes in a single region of
chromosome 11p15. These results provide experimental evidence for the
importance of the proximal portion of the
MUC2 gene in effecting goblet
cell-specific expression, indicate major differences in the regulation
of MUC2 expression in the colon and
the small intestine, and provide insight into goblet cell-specific gene
regulation along the crypt-villus axis of the small intestine.
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METHODS |
Construction of the MUC2-hGH transgene and transgenic
mice. A construct containing bases
2864 to +17
of the human MUC2 gene 5'-flanking region spliced into the 5'-untranslated region
of the hGH gene was prepared using a strategy similar to the one detailed previously for preparing the comparable
MUC2-luciferase construct (13). To
generate an Xba I site at base +17 of
MUC2, the PCR was used with GMUC46 as
target DNA and primers 5'-TAAGGAGCCTGACCAGACTTGCTTCTGGC and
5'-CCATGGTGTCTAGAAGGGGCGGTGTGGG,
embolded bases altered to create the restriction site. The resulting
364-base fragment, which contains the proximal portion of the
MUC2 5'-flanking region, was
digested with Xba I and
Bsm I and ligated to a similarly digested construct containing bases
2864 to +806 of
MUC2 cloned into the
Sal I site of pBluescript. Digestion
with Sal I and
Xba I yielded bases
2864 to +17
of MUC2 for ligation into pOGH
(Nichols Institute, San Juan Capistrano, CA). The portion of the
sequence generated by PCR and the splice points were confirmed by
sequence analysis to ensure the absence of errors. The resulting
transgene containing the hGH structural gene ligated immediately
downstream of bases
2864 to +17 of
MUC2 (Fig.
1) was retrieved by digestion with
Sal I and
EcoR I for transgenic mouse
production. Mice were prepared by microinjecting C57BL/6J × DBA/2J F2 hybrid zygotes with
~500 copies of the transgene according to standard protocols (20).
The resulting founders were bred to C57BL/6J mice to propagate the
lines. The experiments reported here were conducted using F1 or
F2 mice.

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Fig. 1.
MUC2-human growth hormone (hGH)
transgene. The 5' portion of the
MUC2 gene is represented at
top. Exons of
MUC2 structural gene are depicted as
solid blocks and numbered; sequenced portion of 5'-flanking
region contained in clone GMUC46 is also shown. Thin horizontal line
below depicts the transgene. Nar I
site was converted to Sal I site, and
Xba I site was introduced as described
in text. Solid blocks represent the 5 exons of the hGH structural gene.
At bottom, sequence of MUC2
surrounding transcription start site is given. Bent arrow represents
start of transcription, and underlined ATG is initiation codon. Final
sequence is that of the transgene. Bolded bases represent
non-MUC2 sequence. Specifically, 3 bases were altered to create Xba I
site for cloning into pOGH, and sequence from
BamH I site to initiating ATG is part
of the 5'-untranslated region of the hGH gene.
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DNA and RNA extraction and blot
analysis. DNA was isolated from the tips of mouse tails
using proteinase K digestion and
phenol-CH3Cl and
CH3Cl extraction followed by
isopropanol precipitation (3). DNA was digested with
BamH I, electrophoresed, and blotted
to nylon membranes as described previously (12). The
2864 to +17 MUC2 fragment of the transgene was
used as probe. The 520-bp internal BamH I fragment (Fig. 1) was used for
estimation of transgene copy number by comparing the band intensities
of the mouse DNA to standards prepared by digestion of known quantities
of the plasmid used for transgene construction. NIH Image 1.54 software was used for densitometric intensity comparisons of autoradiograms. RNA
was extracted from tissues following homogenization in Tri reagent
using the protocol recommended by the manufacturer (Molecular Research
Center, Cincinnati, OH). For small intestinal RNA extraction, 5-cm
segments were used from the proximal, middle, and distal segments of
the intestine. Colonic RNA was extracted from a 5-cm segment taken from
the middle of the colon. RNA blots were prepared, hybridized, and
washed as previously described. An hGH probe spanning bases
142-823 of the hGH message was prepared using the PCR and plasmid
39384 from the American Type Culture Collection as target DNA. A probe
for the mouse homolog of MUC2 spanning
bases 1573-1912 of the rat sequence (30) was also made using the
PCR with reverse-transcribed mouse colonic RNA as target DNA. This
340-base probe, designated D2, had ~96 and 83% sequence similarity
to the rat and human sequences, respectively. The probe for
glyceraldehyde-3-phosphate dehydrogenase was described previously (15).
RNase protection analysis. Two
constructs were prepared for templates for RNA probe preparation using
the PCR with the transgene-containing plasmid as target DNA. These
constructs both spanned the MUC2-hGH splice junction and both terminated at base 27 of the first intron of
the hGH gene. Construct F1 initiated at base
170 of the
MUC2 5'-flanking sequence and
construct F2 initiated at base
43. The appropriate fragments
were cloned into pBluescript (SK
) and sequenced to ascertain
that point mutations were absent. Pst
I digestion was used to yield templates for antisense probe preparation
using T7 RNA polymerase. Hybridization, digestion, electrophoresis, and
analysis were performed as previously described using 20 µg of total
RNA from distal small intestine or colon (17).
Immunohistochemistry. Mice were
anesthetized using methoxyflurane and killed by cervical dislocation.
Tissues were removed rapidly, rinsed in phosphate-buffered saline,
fixed in Bouin's solution, and embedded in paraffin before the cutting
of 5-µm sections. Rabbit polyclonal anti-hGH (1:500, Dako,
Carpinteria, CA) was used as the primary antiserum in an overnight
incubation as described by Markowitz et al. (29), followed by detection with immunogold silver staining using reagents purchased from Zymed
Laboratories. The silver enhancement procedure was performed either one
or two times, depending on antigen level in a given tissue. Slides were
counterstained using Alcian blue. Double labeling using anti-hGH and
immunogold silver staining followed by rabbit anti-serotonin (1:3,000;
Incstar, Stillwater, MN) and FITC-conjugated pig anti-rabbit IgG (Dako)
was also performed as described by Markowitz et al. (29).
Immunoelectron microscopy. Reagents
for electron microscopy (glutaraldehyde, paraformaldehyde, sodium
cacodylate, uranyl acetate) were purchased from Electron Microscopy
Sciences (Fort Washington, PA). LR Gold resin was obtained from
Polysciences (Warrington, PA). Tissue samples were fixed at room
temperature by immersion in 2% paraformaldehyde and 0.2%
glutaraldehyde buffered with 0.1 M sodium cacodylate (pH 7.20). After 2 h of fixation at room temperature, the samples were washed overnight in
buffer. Tissues were embedded into LR Gold resin, polymerized at
20°C by ultraviolet light (2, 24). Thin (80 nm) sections
were cut on an RMC MT-7 ultramicrotome with a diamond knife and
collected on nickel grids (Polysciences).
For immunogold detection of the hGH reporter, nonspecific binding of
the immunoreagents was prevented by incubating the grids for 30 min on
a drop of blocking buffer: 10 mM Tris buffer (pH 7.40) containing 2%
bovine serum albumin, 0.5% cold-water fish skin gelatin, 0.1% Tween
20, and 500 mM sodium chloride. The grids were then incubated with the
Dako primary antibody (135 µg/ml) in a humid chamber at room
temperature for 12 h, followed by extensive washing with the blocking
buffer (5 times, 5 min each). The antibodies bound to the sections were
detected by goat anti-rabbit
IgG-Au10 (Aurion, Wageninin, The
Netherlands) for 30 min, followed by extensive washing: three times
with blocking buffer (5 min each), three times with 10 mM Tris (pH
7.40) containing 500 mM sodium chloride, and three times with distilled
water. Controls included 1) omission of the primary antibody; 2)
incubation with a nonrelevant antibody, i.e., rabbit anti-porcine IgG
in lieu of the specific antiserum; 3) incubation with a nonrelevant
gold reagent, i.e., goat anti-mouse IgG-Au10 in lieu of the specific
gold reagent; and finally 4) labeling of nontransgenic, control tissues. All of the controls resulted in negligible deposits of colloidal gold over the sections, indicating specific binding of the anti-hGH antibody. The sections were
contrasted with 2% osmium tetroxide for 15 min, washed with distilled
water, and stained with Sato's lead stain. The dried grids were then
coated with 0.25% Formvar (24) and examined and photographed using a
Zeiss 10CR electron microscope at 60 kV.
 |
RESULTS |
Analysis of transgenic mice. DNA
blot analysis indicated the presence of transgene(s) in seven founder
mice. All of these mice were fertile, and all propagated the
transgene(s) to their offspring in an autosomal dominant pattern. One
of the mice, founder 6, apparently incorporated transgenes
in two different genomic locations because its offspring exhibited
different patterns on DNA blot analysis. The two genotypes segregated
in the F1 generation and were
designated lines
6 and
6' (Fig.
2). Thus a total of eight transgenic lines
were produced and available for analysis. All transgenic mice resembled
their normal littermates in appearance.

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Fig. 2.
DNA blot analysis of transgenic lines. Tail DNA from the 8 transgenic
lines and a nontransgenic control (N) were digested with
BamH I and analyzed by blot analysis.
Entire 2864 to +17 MUC2
fragment used for transgene construction was used as a probe.
Quantitation of the 520-bp fragment was performed as described in
METHODS.
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Figure 2 shows a blot containing BamH
I-digested DNA from different lines probed with the
MUC2 portion of the transgene. As indicated on the map shown in Fig. 1, the transgene contains two internal BamH I fragments of 520 and
331 bp. These two fragments were present at various levels in each
transgenic line. Densitometric analysis of the 520-bp fragment
indicated the transgene to be present at 1 to ~32 copies per diploid
genome in heterozygotes (Table 1). The
patterns exhibited by the external
BamH I fragments varied with line.
These fragments hybridize to the region of the probe located between
the Sal I site and the first
BamH I site of the transgene (Fig. 1).
Lines
3, 5,
6', and
8 have only a single copy of
transgene, and they exhibit external fragments of various sizes. This
variation is as expected because the sizes of the external fragments
are dependent on the locations of BamH
I sites in the transgene integration sites of the individual lines.
Concatemeric insertion of multiple copies of transgene would give a
4,200-bp fragment because this is the size of the hGH reporter, which
initiates from a BamH I site, plus the
portion of the MUC2 gene
5'-flanking sequence proximal to the first
BamH I site included within this sequence (Fig. 1). This appears to have occurred with
lines
1 and
2.
Lines
4 and
6 have transgene copy numbers of ~3
and 4, respectively, but do not appear to have undergone concatemeric insertion because they lack the 4,200-bp fragment seen in
lines 1 and
2. The mode of insertion in these two
lines is therefore unknown at present.
Preliminary experiments indicated expression of the hGH reporter gene
in the distal small intestine of several lines. This was examined
systematically using RNA blot analysis, and expression of the 800-base
hGH message was detected in four of the eight lines (Fig.
3). There is a great deal of variation
between the lines that do express, with
line
6 producing only ~2% of the hGH message level found in line
1, which exhibited the highest
level of expression (Table 2). Moreover,
reporter expression levels were not strictly dependent on transgene
copy number. For example, as shown in Table 2,
line
4 expresses approximately four times the reporter when normalized to transgene copy number as does line
1.

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Fig. 3.
RNA blot analysis of transgene expression in all lines. Blot containing
distal small intestine total RNA samples probed for hGH message
expression is shown at top. Blot was
later analyzed using a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
probe to ensure that sample was loaded in each lane.
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Tissue specificity of transgene
expression. To examine the tissue specificity of
transgene expression, RNA was isolated from multiple tissues of 6.5- to
9-wk-old mice and subjected to blot analysis. The four lines that
lacked reporter expression in the distal small intestine (Fig. 3) also
lacked expression in all other tested tissues (not shown). In the other
lines, expression was largely constrained to the intestine. Figure
4 shows autoradiograph sections showing the
hGH reporter message expressed in different tissues from mice of
lines
1, 2,
3, 4,
and 6 and a nontransgenic littermate
as control. Here, the exposure times of the autoradiographs of the
different lines were adjusted according to the expression level within
each line. Relative expression in the middle and distal small intestine
was highest in lines
1, 2,
and 6. The middle small intestine of
line
4 expressed at lower levels than the
distal segment, and the proximal small intestine of all lines exhibited considerably lower expression. Only
line
2 had high relative expression in the
colon. This contrasts with endogenous
MUC2 expression, which is considerably
higher in the colon than in small intestine (Fig. 4 and Table
3). Trace expression was observed in the
stomach and heart of line
1 and the heart and ovary of
line
4. No expression was observed in the
liver, kidney, or brain of any line.

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Fig. 4.
Analysis of transgene expression in various tissues. Mice (6.5-9
wk old) of all lines were analyzed, but only those expressing
transgene, line
3, and a nontransgenic control (N) are
shown. Sexes of individual mice are given (M, male; F, female). Tissues
analyzed are proximal (PSI), middle (MSI), and distal (DSI) small
intestine; colon (Col); stomach (Sto); liver (Liv); kidney (Kid); heart
(Hea); ovary or testes (Ov/T); and brain (Bra). First 6 autoradiogram
swatches show hGH expression. GAPDH message was determined to be
present in all samples, but only results for
line
3 mouse are shown.
Bottom autoradiogram is that of
line
2 mouse probed for endogenous
MUC2 gene expression. Polydispersity
exhibited is characteristic of MUC2
and other mucin transcripts and is caused by shearing forces acting on
large (>15 kb) messages during isolation and subsequent manipulation
(10).
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An important test of the fidelity of a promoter/reporter model to the
endogenous promoter is whether transcription is initiated from the same
position on the promoter in both instances or not. To examine this,
RNase protection assays were performed to map the transcription start
sites of the transgene. Figure 5 depicts an
experiment in which RNA from the distal small intestine and colon of
line
1, 2,
and 4 mice together with a
nontransgenic control was examined. Two separate riboprobes, both
terminating at the same nucleotide in the first intron of the hGH
reporter but initiating at different points in the
MUC2 promoter, were used. Two major fragments of 95 and 98 bases in length and several minor fragments were
obtained with both probes when line
1 or
line
4 distal small intestine RNA was used
(Fig. 5). Line
2 distal small intestine RNA yielded
faint but detectable fragments in the same positions, the expected
result given the low level of transgene expression in this line (Fig.
3). The sizes of these fragments position the major start sites at
bases
27 and
30 of the
MUC2 promoter sequence. This is in
good agreement with the start sites observed for endogenous MUC2 transcription in the human colon,
which exhibited four major start sites between nucleotides
23
and
27 (17). The nontransgenic control RNA produced no protected
fragments. The fact that both probes yielded the same size fragments
provides evidence that the 5'-termini were correctly
approximated, because both probes are protected to the same point on
their 3'-terminus, i.e., to the end of the first exon. When
colonic RNA from line
2 was used, the same two 95- and
98-base fragments were faintly visible with probe F1, but not with
probe F2 (Fig. 5). This is probably due to differential sensitivity of
the assay with the two probes. The other lines did not exhibit bands
with colonic RNA, again the expected result because they express
reporter minimally, if at all, in this tissue (Fig. 4).

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Fig. 5.
Determination of transgene transcription start site. RNase protection
assays were performed as described in text.
A: sequences spanned by the 2 probes
employed in the assays. Complete
MUC2-hGH transgene and region examined
are shown (expanded). Solid blocks represent exons of hGH structural
gene. Note that probes F1 and F2 initiate at different positions in the
5'-flanking region but terminate at the same nucleotide in the
first intron. Single arrowhead represents location of the start of
transcription of the native MUC2 gene in human colon.
B: autoradiograms of RNase protection
assays. Total RNA from distal small intestine and colon of
line
1, 2,
and 4 mice and nontransgenic mice were
tested with both probes. Sizes of protected fragments were determined
by comparison to a DNA sequencing ladder, corrected for 2% difference
in mobility between DNA and RNA (17). Note that similar patterns of
fragments were obtained with both probes. Sequence represented by
region of probes containing intense fragments is shown at
right. Arrowheads mark location of 2 strongest bands obtained with the transgene; dots show major start
sites determined previously for MUC2
transcription using human colonic RNA (17).
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Cell type specificity of transgene
expression. Immunohistochemical analysis was used to
assess the cell type specificity of reporter expression in the various
transgenic lines. Figure
6A shows a
longitudinal section of the distal small intestine of a
line
1 mouse. It is clear that transgene
reporter expression colocalized with the Alcian blue-stained goblet
cells. The silver particles were deposited over the rough endoplasmic
reticulum and Golgi-containing region of the cells as can best be seen
examining villi cut in cross section (Fig.
6B). Close examination of the sections reveals that transgene expression begins deep in the crypt,
far below the crypt-villus junction. Thus transgene expression occurs
as an early event, beginning approximately with differentiation into
the goblet cell lineage. It is also clear that enterocytes, which
constitute the major cell type present in the small intestinal epithelium, do not express transgene. Furthermore, some goblet cells
from line
1 mouse intestine also fail to express
transgene, as can be seen in both Fig.
6A and Fig.
6B. A similar pattern of expression
can be seen with line
4 mice, although the level of
expression in this line is considerably lower (Fig. 6,
C and D). Again, the staining of goblet
cells beginning at positions deep in the crypts is observed. No hGH
reporter expression was observed with nontransgenic mouse intestine
(Fig. 6E). Attempts to localize
transgene expression in line
2 and
line
6 mouse intestine using this technique
were unsuccessful, presumably because hGH protein levels were below the
limits of detection (not shown).

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Fig. 6.
Immunohistochemical analysis of transgene expression in distal small
intestine. hGH reporter expression was visualized using immunogold
silver staining, and mucin-containing goblet cells were identified
using Alcian blue counterstaining. Bars represent 50 µm.
A and
B are from
line
1 mouse,
C and
D are from
line
4 mouse, and
E is from nontransgenic mouse. Arrows
in A and
B represent goblet cells that do not
express detectable levels of reporter. Silver enhancement process was
performed twice on sections from line
4 mice because staining was otherwise
weak.
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The intestinal epithelium contains small numbers of enteroendocrine
cells interspersed among larger numbers of enterocytes and goblet
cells. The immunohistochemical analysis described above suggested
goblet cell-specific expression of reporter but could not rule out the
possibility of transgene expression in enteroendocrine cells, which
occur only infrequently. To examine this, sequential double labeling of
cells in tissue sections was performed using anti-hGH detected with
immunogold silver staining and anti-serotonin detected with
FITC-labeled second antibodies. Serotonin-producing enteroendocrine
cells represent the majority of enteroendocrine cells produced in the
mouse distal small intestine (32). Examination of the double-labeled
sections revealed segregation of the labels into different cell types;
i.e., coexpression of the two labels in the same cells was not
observed. Illustrative of this experiment, Fig.
7 shows three serotonin-producing
enteroendocrine cells that lack reporter expression. Thus these results
indicate that detectable levels of transgene reporter are not expressed
in serotonin-producing enteroendocrine cells.

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Fig. 7.
Sequential immunodetection of hGH and serotonin in distal small
intestine of line
1 mouse. Tissue sections were
processed to localize hGH expression by immunogold silver staining and
serotonin expression using FITC-labeled second antibodies. Bright-field
micrograph showing hGH expression is shown in
A, with its corresponding fluorescent
micrograph showing serotonin expression shown in
B. Arrows depict location of
serotonin-producing cells on bright-field micrograph. Bar represents 50 µm.
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Ultrastructural localization of transgene
expression. We expanded our examination of transgene
expression in the intestine to the ultrastructural level using
immunoelectron microscopy. These studies confirmed and extended our
light microscopic observations. Electron microscopy revealed no
apparent ultrastructural differences in the distal small intestinal
mucosa of control vs. transgenic animals (Fig.
8,
A-C).
In control animals, no hGH-reactive cells were found.
Line
1 distal small intestinal mucosa,
however, contained a subset of goblet cells with hGH-reactive,
electron-dense secretory granules (Figs. 8,
C and
D, and 9).
No other cells, including Paneth cells (Fig.
8B), enteroendocrine cells, or
enterocytes, were noted that contained detectable hGH reporter. Closer
examination of the ultrastuctural characteristics of the goblet cells
revealed that the hGH-reactive, small, electron-dense granules were
located within the mucus globules (Figs. 8,
C and
D, and 9,
A-D).
The mucus globules themselves did not contain any hGH-reactive
material. These ultrastructural features indicate that the
hGH-expressing goblet cells are identical to the so-called granular
goblet cells described earlier by Cheng (7). No intracellular
organelles, including the endoplasmic reticulum, Golgi apparatus, or
mucus globules, contained any hGH reactivity in common goblet cells, which lack the dense granules (Fig.
8A), the identifying feature of
granular goblet cells. Whereas hGH-reactive material was not detected
in the endoplasmic reticulum of granular goblet cells, it was clearly
seen in the trans-Golgi network, where the label was associated with
vesicles of different sizes (Fig. 9, A
and B). The detection of reporter in
the Golgi but not the endoplasmic reticulum probably indicates a higher
reporter concentration in the former organelle, possibly as a result of
a packaging process. Label was also found in the presumably nascent
dense granules associated with the trans-Golgi (Fig.
9C). In the mature granules the label was seen both at the granular surface and in the matrix. Secretion, i.e., exocytotic release of the granules, was observed. However, no hGH-reactive material was seen in the mucus, possibly because of degradation or dilution of the epitope in the intestinal lumen.

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Fig. 8.
Expression of transgene is restricted to granular goblet cells. LR
Gold-embedded sections of proximal small intestine of
line
1 mouse were immunolabeled to detect
hGH transgene protein. No expression was detected in conventional
goblet cells (A) or in Paneth cells
(B), whereas granular goblet cells
expressed the protein, which is localized in dense granules
(C). Boxed area in
C is enlarged in
D. Bars are 1 µm.
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Fig. 9.
Localization of transgene in granular goblet cells. LR Gold-embedded
sections of proximal small intestine of
line
1 mouse were immunolabeled to detect
transgene. Whereas no hGH-reactive material is seen in endoplasmic
reticulum (A and
B), it is associated with membranes
of vesicles in the trans-Golgi (B)
and with the dense core of granules within condensing vesicles
(B). In mature dense granules, label
is associated with both granular periphery and inner matrix
(C and
D). Bars in
A and
B are 1 µm, whereas in
C and
D bars are 0.25 µm.
|
|
 |
DISCUSSION |
Highly glycosylated, polymeric, secreted mucins such as
MUC2 serve to coat and protect the
gastrointestinal, reproductive, and respiratory tracts. Given their
role in protection, large size, and the complex processes required for
their biosynthesis, it is not surprising that alterations in mucins,
and their levels of expression, are associated with several diseases.
In certain cancers, mucin oligosaccharides are altered in several ways,
including a reduction in number, shortening, the exposure of normally
cryptic inner chain residues, and the expression of novel carbohydrate structures (21, 23). The levels and types of mucins expressed are
altered in cancers as well. For example, human cancers, especially colon cancers, differ widely in the levels of
MUC2 they express (18). Most colon
cancers express only low levels of
MUC2 compared with normal colon, but a
class known as mucinous colon cancers expresses high levels of
MUC2, suggesting that they follow a
different mutational pathway to malignancy (19). Several diseases of
the airways are associated with altered mucin gene expression as well (22, 33). These include cystic fibrosis, in which
Pseudomonas infection appears to
induce MUC2 expression, perhaps
contributing to airway obstruction (26). This association of altered
mucins with disease has led us to examine in detail the structure and function of mucins and the factors that control their expression.
We and others have characterized MUC2
gene expression in vitro using transient transfections of cultured
cells with promoter/reporter constructs and nuclear protein binding
studies. This work has identified a CACCC motif located between bases
88 and
80 of the MUC2
reporter that binds Sp1 and related factors and that appears to be
important functionally because its presence establishes a low level of
transcription in all tested cells whether they express
MUC2 or not (13). We have also
identified bases
228 to
171 as possibly being important
in conferring cell-type specificity on the
MUC2 promoter and a nuclear
factor-
B site located between bases
1452 and
1441 that
participates in the induction of MUC2 by Pseudomonas (13, 26, 27). It is
important to examine the expression of
MUC2 in vivo, however, because tissue
culture systems lack high-level expression and authentically
differentiated cell types, essential components of cell type-specific
gene expression. Previous studies using transgenic systems have shed
considerable insight into the factors that control gene regulation in
enterocytes, enteroendocrine cells, and Paneth cells (4, 11, 25, 36). In this work we extend the examination of intestinal gene expression in
vivo to the fourth cell lineage, i.e., goblet cells.
Tissue-specific expression of the MUC2-hGH
transgene. The
MUC2-hGH transgene is expressed at
high relative levels in the distal small intestine of four of the eight
lines obtained (Figs. 3 and 4). High levels of reporter expression in
the middle small intestine are also observed in three lines. On the
contrary, very low expression levels were observed in the proximal
small intestine and significant expression in the colon was observed
only in line
4. This contrasts with endogenous
MUC2 gene expression, which follows an
increasing gradient along the horizontal axis of the intestine (Table
3). Also, significant transgene expression was not observed in the non-MUC2-expressing tissues examined.
This complex pattern of expression indicates that many elements
function in the control of MUC2 levels
in tissues, as has been observed in experiments examining the promoters
of other intestinal genes in transgenic mice (9, 34, 38).
Transgene expression in the distal small intestine indicates that
elements required for expression in this tissue are located between
bases
2864 and +17 of the MUC2
5'-flanking sequence. Clearly, however, this expression is
influenced by the local genetic environment dictated by insertion site
because four of the eight lines expressed no detectable levels of
reporter and in the other lines there was wide variation in the level
of reporter expressed when normalized to copy number of transgene
incorporated (Table 2). Marked integration site dependence for
transgene expression was observed using bases
201 to +54 of the
Si promoter but not when the
8500 to +54 sequence was used (38). This result was attributed
to the ability of the region between bases
8500 and
201
to insulate the transgene from surrounding chromatin effects. In this
regard, it is interesting to note that
MUC2 resides within a 400-kb region of
chromosome 11p15.5 that contains at least three other secretory mucin
genes (31). There may be regions within this
"MUC cluster" that have
insulator activity or even that function in the regulation of several
mucin genes. The lack of even moderate levels of reporter expression in
all tested non-MUC2-expressing tissues
suggests the possibility that at least one silencer located between
bases
2864 and +17 of the MUC2
5'-flanking sequence is an important component in achieving
tissue-specific regulation.
The fact that reporter is expressed in the colon in only one transgenic
line is especially surprising. This result lies in stark contrast to
what is observed in the small intestine of the same animals and
strongly suggests that different combinations of nuclear factors
regulate MUC2 expression in the small
intestine and colon. One possibility is that elements found elsewhere
than on the
2864 to +17 region of
MUC2 are utilized by colon-specific factors to produce high-level expression. Possibilities include sequences found upstream of base
2864, in introns, or even in the 3'-flanking sequence. Experiments involving other intestinal genes suggest that multiple enhancers and silencers function in concert
to effect tissue-specific expression (11, 36). The similar locations of
the transcription start sites of the transgene in mouse intestine (Fig.
5) and endogenous MUC2 in human colon indicate a common utilization of basal transcription factors by the
MUC2 promoter in human and mouse
intestinal cells, an expected result because several hundred bases of
the proximal portion of the MUC2
promoter are conserved between human and mouse (14). The observation
that one of the lines did express reporter at approximately equal
levels in the colon and small intestine is again suggestive of the
notion that the local genomic environment plays an important role in
MUC2 gene regulation, and perhaps this effect is more important for colonic expression than for small intestinal expression.
Cell type-specific expression of the MUC2-hGH
transgene. Imunohistochemical analysis indicates that
the hGH reporter is expressed in the goblet cells of the small
intestine (Fig. 6). Colocalization of hGH antigen with Alcian
blue-stained mucin goblets occurs deep in the crypts, indicating a
close temporal relationship between the initiation of transgene
expression and the development of the goblet cell phenotype. The
message for human MUC2 is also expressed deep in the crypts (1, 5). Thus transgene expression initiates in a similar early position along the crypt-villus axis as
does endogenous MUC2 expression. This
implies that elements required for this important aspect of
MUC2 gene regulation appear to be
located between bases
2864 and +17. As the goblet cells migrate
past the crypt-villi junction, immunohistochemical staining of reporter
becomes less pronounced and some Alcian blue-staining cells are clearly
negative for reporter expression. This is in apparent contradiction to
previous in situ hybridization studies that indicate that
MUC2 expression persists the entire
lifetime of goblet cells in the human small intestine (1, 5). It is
possible, however, that this apparent discrepancy results from differences between the translational efficiencies of the messages and/or the processing and secretion of hGH and
MUC2 rather than reflecting true
differences in transcriptional regulation. It is also possible,
however, that regulatory sequences required for continued high-level
reporter expression in the villi are found elsewhere than on the
portion of the MUC2 5'-flanking
sequence used in transgene construction.
The absence of reporter expression in small intestinal epithelial cells
other than goblet cells suggests either that silencers that suppress
MUC2 expression in inappropriate
epithelial cell types reside in the
2864 to +17 region or that
goblet cell-specific enhancers reside in this region. The cell type
specificity observed is analogous to similar transgenic models using
the cryptidin 2 gene promoter and the glucagon promoter, which exhibit
expression limited to Paneth cells and enteroendocrine cells of the
adult mouse small intestine, respectively (4, 25). It contrasts, however, with transgenic models using the promoters of enterocyte specific genes Si,
Fabpi, and
Fabpl, in which reporter was detected in most epithelial cell types (11, 36). These results suggest fundamental differences between gene regulation in enterocytes and the
other less-abundant cell types of the intestinal epithelium.
Previous electron microscopic analysis of the mouse small intestine
revealed the presence of two types of mature goblet cells, designated
common and granular mucus cells (7). These cells are similar in size
and shape and are distinguished by the presence of electron-dense
granules of 200-500 nM that are located within the mucus globules
of granular mucus (goblet) cells. Approximately 23% of the goblet
cells of the distal small intestinal crypts are granular goblet cells,
the remainder being of the common variety. As the cells migrate to the
villi, granular goblet cells become much less common without any
evidence of a degenerative process, leading to the hypothesis that they
become common goblet cells (7). In this study, we examined hGH reporter
expression by immunoelectron microscopy (Fig. 8). Here, we found
reporter expression only in granular goblet cells. Other cell types of
the small intestinal epithelium, including common goblet cells, were
not labeled. The limitation of hGH reporter protein expression to a
subclass of goblet cells raises several interesting possibilities.
First, transgene expression could actually be limited to granular
goblet cells; i.e., elements found elsewhere than on the
2864 to
+17 region may be required for expression in common goblet cells. This
hypothesis is discredited considerably by the large numbers of villi
goblet cells observed labeled by anti-hGH in typical histological
sections. It is also possible that granular goblet cells differ
metabolically from common goblet cells. This could lead to the
detection of hGH by immunoelectron microscopy only in granular goblet
cells, with both classes expressing antigen when examined using
conventional immunohistochemistry, the more sensitive of the two
techniques. Rapid secretion of the foreign protein hGH vis-à-vis
MUC2, which is typically stored by
intestinal goblet cells before release, may be a factor in this regard
as well. A study utilizing transgenic mice harboring an
Fabpl-hGH transgene is especially
relevant to our observations here (37). In this earlier study, similar
immunoelectron microscopic techniques were used and expression was
observed in enterocytes, enteroendocrine cells, Paneth cells, and
granular goblet cells. Common goblet cells were the only small
intestinal epithelial cell type without hGH antigen detectable by
immunoelectron microscopy in the
Fabpl-hGH mice (37). This work using a
promoter with broad specificity for expression in small intestinal
epithelial cells suggests that some unique feature of common goblet
cells prevents the immunoelectron microscopic detection of hGH antigen
in this cell type. Also, this earlier study indicates that hGH antigen
is detectable in enterocytes, Paneth cells, and enteroendocrine cells.
This suggests further that the inability to detect hGH by
immunoelectron microscopy in these cell types in the
MUC2-hGH mice is a result of cell
type-specific expression. Additional work will be necessary to
investigate the interesting observation that common goblet cells lack
hGH expression detectable by immunoelectron microscopy.
 |
ACKNOWLEDGEMENTS |
This work was supported by the Department of Veterans Affairs
Medical Research Service, a VA-DOD Merit Research Award, and by the
University of California at San Francisco Cystic Fibrosis Core
Facility, funded by National Institute of Diabetes and Digestive and
Kidney Diseases Grant DK-47766 to C. J. Epstein.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. R. Gum, Jr.,
Gastrointestinal Research Laboratory (151M2), 4150 Clement St., San
Francisco, CA 94121 (E-mail: jgum{at}maelstrom.ucsf.edu).
Received 27 August 1998; accepted in final form 17 November 1998.
 |
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