Murine colonic mucosa hyperproliferation. I. Elevated CFTR
expression and enhanced cAMP-dependent Cl
secretion
Shahid
Umar,
Jason
Scott,
Joseph H.
Sellin,
William P.
Dubinsky, and
Andrew P.
Morris
Department of Integrative Biology, Pharmacology and Physiology,
and Department of Internal Medicine, Division of Gastroenterology,
Hepatology and Nutrition, The University of Texas Health Science Center
at Houston, Medical School, Houston, Texas 77030
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ABSTRACT |
Fluid transport in the large intestine is mediated by the
cystic fibrosis gene product and cAMP-dependent anion channel cystic fibrosis transmembrane conductance regulator (CFTR). cAMP-mediated Cl
secretion by gastrointestinal cell lines
in vitro has been positively correlated with the insertion of CFTR
into the apical membrane of differentiated senescent colonocytes and
negatively correlated with the failure of CFTR to insert into the
plasma membrane of their undifferentiated proliferating counterparts.
In native tissues, this relationship remains unresolved. We
demonstrate, in a transmissible murine colonic hyperplasia (TMCH)
model, that (8-fold) colonocyte proliferation was accompanied by
increased cellular CFTR mRNA and protein expression (8.3- and 2.4-fold,
respectively) and enhanced mucosal cAMP-dependent Cl
secretion (2.3-fold). By immunofluorescence microscopy, cellular CFTR
expression was restricted to the apical pole of cells at the base of
the epithelial crypt. In contrast, increased cellular proliferation in
vivo led to increases in both the cellular level and the total number
of cells expressing this anion channel, with cellular CFTR staining
extending into the crypt neck region. Hyperproliferating colonocytes
accumulated large amounts of CFTR in apically oriented subcellular
perinuclear compartments. This novel mode of CFTR regulation may
explain why high endogenous levels of cellular CFTR mRNA and protein
within the TMCH epithelium were not matched with larger increases in
transmucosal CFTR Cl
current.
cystic fibrosis transmembrane conductance regulator; regulation; location; mRNA; protein
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INTRODUCTION |
THE FUNCTIONAL EXPRESSION of the cystic fibrosis (CF)
gene product, cystic fibrosis transmembrane conductance regulator
(CFTR), is pivotal for intestinal Cl
secretion
elicited by neurohormonal agonists acting both through cAMP and
Ca2+ (3). CFTR Cl
channels open after
phosphorylation by protein kinase A (2). The polarized expression of
CFTR within the apical membrane (13) is thereby believed to control the
exit of cellular Cl
into the intestinal lumen.
Transcellular Cl
movement, coupled to paracellular
Na+ egress through the epithelial tight junction,
constitutes the ionic basis for NaCl secretion and fluid production in
the intestine and many other epithelial tissues (9). Homozygous
mutations in the CFTR genome have been found to either eliminate or
severely curtail this apical membrane cAMP-regulated
Cl
permeability pathway in CF epithelia. Extensive
characterization of these genomic changes has revealed that mutations
manifest their effects at multiple levels within the cell. In general
they can be categorized as causing either loss of or diminished CFTR expression, inhibition in the cellular processing/targeting of CFTR
protein, or attenuations in anion channel function (20). All of these
cellular effects result in the same pathophysiological consequence: a
lack of functional CFTR within the apical membrane of intestinal
epithelial cells, which is believed to be the basic cellular defect
underlying the clinical manifestations of CF (20).
The localization of CFTR mRNA and protein in gastrointestinal cell
lines and the digestive tract of normal and transgenic CF mice has been
correlated with the ability of individual epithelial cells/glands to
secrete Cl
in response to cAMP agonists. The
crypt-villus and crypt-surface axes of the small and large intestines,
respectively, provide some of the most startling examples of this
phenomenon. High levels of CFTR mRNA and protein expression within the
immature cell populations of the crypt taper off to lower or
nonexistent levels in the more mature villi/luminal surface regions (7,
12). This CFTR distribution thereby identifies the intestinal crypt as
the primary site of fluid secretion. However, given the proposed
importance of the immature intestinal crypt cells to tissue generated
cAMP-dependent Cl
transport, little is known about
how CFTR expression is regulated in vivo. To address this question, we
employed an animal model in which changes in CFTR-dependent anion
transport were investigated in native colon undergoing enhanced
epithelial proliferation. Transmissible murine colonic hyperplasia
(TMCH), characterized by significant epithelial cell proliferation
within the epithelial mucosa of the descending colon, develops in mice
infected with Citrobacter rodentium (4). In contrast to
previous in vitro findings, proliferation in vivo led to an
increase in both cellular CFTR anion channel expression and net mucosal
cAMP-dependent Cl
secretion. The subcellular
distribution of endogenous CFTR was also changed; CFTR accumulated in
intracellular structures removed from the apical plasma membrane. This
may represent a means by which the hyperproliferative epithelium can
downregulate increases in the functional expression of this anion
channel that are potentially deleterious for the cell.
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METHODS |
Antibodies
Antiserum against the CFTR protein was raised against purified bovine
CFTR. A second affinity-purified murine monoclonal antibody (IgM
subclass) made against whole-molecule human CFTR (TAM18) was purchased
from Labvision (Fremont, CA). BODIPY-conjugated goat anti-bovine and
FITC-conjugated goat anti-rabbit secondary antibodies were purchased
from Molecular Probes (Eugene, OR). Cy2-conjugated IgG goat anti-mouse
IgM heavy-chain isoform-specific secondary antibody and unlabeled fab
anti-IgM fragment were kindly donated by Dr. W. Stegeman (Jackson
Laboratories). Goat polyclonal anti-IgA antibody was purchased from
Sigma Immunochemicals (St. Louis, MO). Finally, affinity-purified
murine IgM panleukocyte CD15-specific control primary monoclonal
antibody was also purchased from Labvision.
Development of a Model for Hyperplasia
TMCH was developed in male Swiss Webster mice (15- 20 g; Harlan
Sprague Dawley, Houston, TX) by oral inoculation with 16-h culture of
Citrobacter freundii (biotype 4280, ATCC) (4). Age-matched control mice received sterile culture medium only. Biotype 4280 is a
unique mouse-specific hybrid Citrobacterium strain (also known
as Citrobacter rodentium) that adheres to mature surface colonocytes within the distal colon to induce histopathological changes
known as attaching and effacing lesions (4). Adherent bacteria were
assayed using RT-PCR for bacterial intimin in whole tissue extracts (1,
16) and were found to be absent during the period of most pronounced
mucosal hyperproliferation when changes in cellular CFTR anion channel
abundance and ion transport were recorded (day 12 after Citrobacter inoculation; data not shown).
To determine gross morphological changes within the colonic mucosa,
animals were killed by cervical dislocation and their distal colons
were removed and flushed with HEPES-buffered saline (in mM: 140 NaCl,
4.7 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES, pH 7.2). Tissues were then embedded in optimum cutting temperature compound (Miles, IN), cryopreserved in liquid
N2, and then sectioned and stained with hematoxylin and
eosin. Goblet cell number was analyzed in the 5-µm-thick sections by
counting the unstained translucent mucin-containing vacuoles.
Photographic slides were digitized at high resolution (2,400 DPI), and
areas were measured using Universal Imaging's Metamorph Software (West Chester, PA). Estimates of inflammatory cell number were made by
counting the total number of cells within the lamina propria. To
estimate the degree of mucosal hyperproliferation, both control and
infected animals were given intraperitoneal injections (160 mg/kg body
wt) of 5'-bromodeoxyuridine (BrdU; Sigma) 1 h before death to
label the S-phase cells. Colons were divided into proximal and distal
sections, attached to a paddle, and immersed in Ca2+-free
standard Krebs-buffered saline (in mmol/l: 107 NaCl, 4.5 KCl, 0.2 NaH2PO4, 1.8 Na2HPO4,
10 glucose, and 10 EDTA) at 37°C for 10-20 min, gassed with
5% CO2/95% O2. Individual crypt units were
then separated from the submucosa/musculature by intermittent (30-s)
vibration into ice-cold potassium gluconate-HEPES saline (in mmol/l:
100 potassium gluconate, 20 NaCl, 1.25 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, and 5 sodium pyruvate) and
0.1% BSA. Crypt suspensions were then deposited (1,200 rpm for 1 min)
onto poly-L-lysine-coated microscope slides using a
Cytospin cell preparation system (Shandon, Pittsburgh, PA). For
detection of incorporated BrdU in S-phase cells, isolated crypts were
incubated with a 1:1,000 dilution of affinity-purified goat anti-BrdU
antibody at 4°C overnight after blocking of nonspecific
protein-binding sites with PBS containing 2% BSA, 0.2% nonfat dry
milk, and 0.3% Triton X-100. Bound anti-BrdU antibody was subsequently
visualized by immunofluorescence staining with BODIPY
FL-conjugated donkey anti-goat IgG antibody. Apoptotic index was
measured after incorporation of fluorescein-labeled dUTP into cellular
DNA by terminal deoxynucleotidyltransferase (TdT) TUNEL assay
(TdT-mediated dUTP nick end labeling). Both labels were detected and
quantified by fluorescence microscopy.
Ussing Chamber Studies
The effects of the cAMP-elevating fluid secretory agonist forskolin on
CFTR-mediated ion transport in normal and hyperproliferative mouse
colon was studied by monitoring short-circuit current
(Isc) responses by automatic voltage clamp.
Unstriped 1.5-cm colonic mucosal sheets encompassing the cecal
(region 1) and rectal (region 4) colonic boundaries
were placed into custom-designed Ussing chambers. All experiments were
carried out at 37°C; standard Krebs-bicarbonate-Ringer solutions
were gassed with 95%O2 -5% CO2 by airlift
circulators. Transepithelial potential difference was clamped to 0, and
the Isc was continuously displayed on a pen
recorder. Transepithelial resistance was calculated from the magnitude
of the current deflections in response to a voltage pulse imposed on
short-circuited cell sheets every 60 s with a duration of 0.5 s (14).
Northern Blot Analysis and RT-PCR
Total or poly(A)+ mRNA was isolated from whole normal and
Citrobacter-infected distal colon as well as from purified
crypts using TRIzol reagent (GIBCO BRL, Grand Island, NY) or the micro Fast Track kit (Invitrogen, San Diego, CA) according to the
manufacturers' instructions. For Northern blot analysis, each
preparation [2.5 µg poly(A)+ mRNA, 10 µg total
RNA] was denatured and fractionated on a 1% agarose gel
containing formaldehyde. RNA was then transferred to a GeneScreen Plus
nylon membrane (DuPont NEN), and the blot was hybridized at 60°C in
10% dextran sulfate, 1 M NaCl, 1% SDS, and 100 µg/ml denatured
salmon testes DNA, with the use of a [
-32P]dCTP-labeled probe encompassing the R domain of
CFTR (bases 1,773-2,654, 2 × 106 cpm/ml) and
subsequently with a probe against glyceraldehyde 3-phosphate
dehydrogenase (GAPDH; bases 163-608, 1 × 106
cpm/ml). The latter signal was used to normalize the mRNA in each lane.
The probe for CFTR detection was generated by PCR of full-length CFTR
cDNA, and the GAPDH probe was generated by RT-PCR from mouse colonic
RNA (13). Both were confirmed by oligonucleotide sequencing before
random primed labeling.
Tissue Preparation for Western Blot Analysis
Swiss Webster mice were killed by cervical dislocation
after 0, 1, 3, 6, 9, 12, and 15 days after Citrobacter
inoculation. Crude homogenates were prepared from the whole distal
colon and isolated crypts from three normal and
Citrobacter-infected animals were prepared for each
experimental observation by homogenization in detergent
containing buffer (in mM: 50 Tris · HCl, 250 sucrose, 2 EDTA, 1 EGTA, pH 7.5, 10 2-mercaptoethanol, and 0.5% Triton X-100,
plus protease inhibitors) followed by a low-speed spin (15,000 g for 15 min). The clear supernatant was saved as total cell
extract. Protein concentration was measured before electrophoresis. Mouse brain homogenates and purified bovine tracheal CFTR acted as
positive control for the CFTR immunoblotting assay. The total cell
extract (30 µg protein/lane) was subjected to 10% SDS-PAGE and
electrotransferred to nitrocellulose membranes. The efficiency of
electrotransfer was checked by backstaining gels with Coomassie blue
and/or by reversible staining of the electrotransferred protein directly on the nitrocellulose membrane with ponceau S solution. No
variability in transfer was noted. Destained membranes were blocked
with 5% nonfat dried milk in 20 mM Tris · HCl and
137 mM NaCl, pH 7.5 (TBS) for 1 h at room temperature and then
overnight at 4°C. Immunoantigenicity was detected by incubating the
membranes for 2 h with either CFTR polyclonal or monoclonal antibody
(0.5-1.0 µg/ml in TBS containing 0.1% Tween 20). After washing,
membranes were incubated with horseradish peroxide-conjugated goat
anti-rabbit IgG (Sigma) or goat anti-mouse IgM (Zymed, San Francisco,
CA) secondary antibodies and developed using the ECL detection system (Amersham, Arlington Heights, IL) according to the manufacturer's instructions.
Immunofluorescence Localization Studies
Region 4 (late distal colon) from normal and
Citrobacter-infected animals were attached to paddles and
immersed in Ca2+-free standard Krebs-buffered saline (in
mmol/l: 107 NaCl, 4.5 KCl, 0.2 NaH2PO4, 1.8 Na2HPO4, 10 glucose, and 10 EDTA) at 37°C for 10-20 min, gassed with 5% CO2/95%O2.
The crypts were then separated from the surrounding connective
tissue/muscle layers by mechanical vibration for 30 s into ice-cold KCl
HEPES saline (in mmol/l: 100 potassium gluconate, 20 NaCl, 1.25 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, and 5 sodium pyruvate) and 0.1% BSA, resembling the intracellular medium.
Freshly isolated or carbowax-preserved (3% polyoxyethylene-29%
denatured ethanol-2% isopropanol; Cytospin collection fluid) crypt
suspensions were then deposited (1,200 rpm for 1 min) onto
poly-L-lysine-coated microscope slides using the Cytospin
cell preparation system. Immunolocalization studies were carried out by
permeabilizing the crypts for 1-3 h at room temperature with 3%
sodium deoxycholate (wt/vol in PBS) in a humidified chamber. An
extended period of detergent permeabilization and extraction was found
to greatly facilitate antibody specificity and reduce background in
preserved crypts. Crypts were stained for CFTR using either
commercially available mouse anti-human monoclonal antibody or rabbit
anti-bovine CFTR polyclonal antibody diluted in blocking solution at
1:200 and 1:100, respectively. After incubation at room temperature for
1 h or at 4°C overnight, the slides were washed and incubated with
either affinity-purified Cy2 conjugated goat anti-mouse IgM heavy-chain
isoform-specific secondary antibody or goat anti-rabbit secondary
antibody conjugated with FITC diluted in blocking solution at 1:500 for
1 h at room temperature or overnight at 4°C. Between washes, slides
were washed for 30 min in PBS containing 1% BSA. Control slides were
incubated without the primary antibody or with affinity-purified murine IgM CD15 panleukocyte-specific monoclonal antibody. Positive
identification of low endogenous levels of mouse crypt IgM was
accomplished using a goat anti-murine IgM antibody. Further controls
involving preincubation of the crypts with unlabeled goat anti-mouse
IgA and the F(ab)2 fragments of the goat anti-mouse IgM
µ-chain-specific antibody were also performed. Fluorescence was
viewed using a Noran confocal laser scanning microscope (CLSM, Noran
Instruments, Middleton, WI) equipped with an argon laser and
appropriate optics and filter modules for fluorophore detection.
Digital images on the CLSM were obtained at ×400, ×800, and
×1,200 using a high numerical aperture lens (Nikon ×40, 1.4 N/A). A z-axis motor attached to the inverted microscope stage
was calibrated to move the plane of focus in 0.4-µm steps through the
sample. Eight or sixteen-bit images collected at 512 × 480 resolution were then stored on a mass storage device (removable
rewritable optical hard disk) and volumetrically reconstructed using
the Image-1/Metamorph 3-D software module (Universal Imaging, West
Chester, PA).
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RESULTS |
Given the marked differences in CFTR mRNA abundance along the in
vivo crypt base-to-surface axis, it is possible that the expression of functional CFTR protein is regulated by or in conjunction with the proliferative status of intestinal epithelial cells (12). This
would represent a clear difference with in vitro data in which CFTR
expression was not affected by cellular proliferatory status (reviewed in Ref. 12). We therefore investigated changes in CFTR
abundance and functional expression in the TMCH model.
Transmissible Murine Colonic Hyperplasia Develops in Swiss Webster
Mice
Establishment of model.
Citrobacter infection induced a predictable and reproducible
hyperplasia in the mouse colon (48 out of 48 animals exhibited dramatic
effects). Grossly detectable thickening and rigidity of the distal half
of the colon was first observed around day 6 after infection
(see Refs. 1 and 17). These changes were occasionally observed in
middle/proximal regions but were never as severe. To more accurately
characterize this phenomenon, the entire colon, encompassing cecal and
rectal boundaries, was separated into four consecutive ~1.5-cm
segments, with segment 1 being the most proximal. After
12-15 days of Citrobacter infection, gross changes were
most evident distally (region 4, 98%; region 3, 74%; region 2, 20%; and region 1, 0%; n = 156 mice, shown as percentages of animals exhibiting 1.5-fold increase in
normal mucosal thickness). The cecum was empty and contracted, but in
no instance was the mucosa grossly thickened. Transverse fixed and
hematoxylin and eosin-stained sections revealed that crypt length in
the descending colon increased more than twofold (region 4);
the crypt length in uninfected animals (220 ± 19 µm) was less than
half that in day 12 post-Citrobacter-infected animals
(460 ± 46 µm, see Fig. 1). TMCH was not
associated with an increased goblet cell number (Fig. 1). In fact, the
average goblet cell area/crypt decreased from 34% to 18% (n = 6 whole mount slides from 6 animals). We did not find significantly
more mesenchymal cells within the submucosal cell layers. Estimates
from eight sections of distal colon region 4 from both control
and day 12 post-Citrobacter-infected mice revealed
similar counts in both samples (14 ± 6 and 19 ± 4 cell nuclei/100
µm2, respectively). The lack of any change in lamina
propria cell number confirms the findings of Barthold and colleagues
(4), who have demonstrated that TMCH in Swiss Webster mice was not accompanied by a significant inflammatory axis (characterized as
recruitment of mononuclear leukocytes/neutrophils into the mucosal and
lamina propria regions). Regions 3 and 4, encompassing the whole of the distal colon, were combined and used for all of the
following biochemical and immunological assays.

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Fig. 1.
Citrobacter-induced hyperplasia. Normal (N) and day 12 Citrobacter-infected (H) mucosa showing thin (5 µm) cryosections
of mouse distal colon fixed and incubated with hematoxylin and eosin
stain magnified at ×4 (A) and higher magnification view
(×20) of purified isolated crypts (described in
METHODS) from similar tissues (B). Mucosal
overgrowth in whole sections of Citrobacter-infected mucosa
correlated with a dramatic increase in isolated crypt length. Bars *1
and *2 = 500 µm and 250 µm, respectively.
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Proliferative and apoptotic indices in normal and
Citrobacter-infected distal colon.
Isolated distal colonic crypts from day 12 post-Citrobacter-inoculated mice contained more
BrdU-labeled cells than controls. Proliferative index (number of
BrdU-labeled S-phase cells/total number of cells in the crypt unit × 100) increased eightfold and was significantly different from
control mucosa (n = 60 crypts/4 mice; P < 0.001, Student's t-test; Fig.
2A). Apoptotic index, a measure of
the fraction of cells undergoing apoptosis, detected by TUNEL
assay/crypt (0.08 ± 0.04 vs. 0.12 ± 0.04, Citrobacter-infected vs. normal mice, means ± SD; n = 60 crypts/4 mice) was not significantly different in crypts taken from
uninfected and infected animals (P < 0.01, Student's
t-test; Fig. 2B) A few cells within the upper reaches
of the crypt were labeled in both instances. Mucosal inflammation within the gut mucosa is characterized by excessive colonocyte apoptosis (19). Our results confirmed that similar conditions were
absent at day 12 after Citrobacter infection. The lack
of counterbalancing programmed cell death in the presence of elevated rates of mitosis within the crypt therefore provides a mechanism for
mucosal hyperplasia in Citrobacter-infected mice.

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Fig. 2.
Measurement of crypt cell dynamics. Proliferatory index (PI; A)
and apoptotic index (AI; B) for distal colonic crypts isolated
from normal (N) and day 12 post-Citrobacter-infected
(H) mice. PI (see METHODS) was elevated more than eightfold
during Citrobacter infection, with higher number of mitotic
cells being found within lower regions of elongated crypts. AI (see
METHODS) did not change during this period. Values are
means ± SD from 3 independent experiments.
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Mucosal Hyperproliferation Affects cAMP-Mediated
Cl
Secretion
To examine the effect of colonic mucosal hyperproliferation on
CFTR-dependent fluid secretion, we measured Cl
secretion in response to elevated cellular cAMP levels in both normal
and day 12 post-Citrobacter-inoculated mice.
Short-circuited secretory currents (Isec) were
recorded across the four 1.5-cm colonic regions encompassing the cecal
(region 1) through rectal (region 4) colonic boundaries.
Bilateral addition of the cAMP-generating agonist forskolin (10 µM)
to normal mucosa elicited between +14 and +30 µA/cm2 of
Isec (region 1 to region 4,
respectively; n = 6; Fig. 3). This
current was abolished by the removal of bath Cl
(n = 6; Table 1). In contrast,
forskolin addition to Citrobacter-infected mouse colonic
segments elicited a significantly larger Isec
(P < 0.001) that averaged +67 ± 17 µA/cm2
within distal regions 3 and 4 (n = 6; Fig. 3).
In some mice (3 out of 6), a smaller increase in
Isec in region 2 was observed. However,
none exhibited enhanced forskolin Isec across the
most proximal colonic segments (n = 6). All secretory currents
were abolished by the serosal addition of 300 µM furosemide
(n = 12; Table 1). Measurements of tissue resistance
[estimated from Isc and open-circuit
potential difference by Ohm's law, where resistance (R) = voltage (V)/current (I)] across the four
consecutive segments of normal and hyperproliferative colonic mucosa
were very similar. Correspondingly, tissue conductance
(Gt = 1/R = I/V) was not
statistically different (P < 0.01; see Table 1 for
individual values). Mucosal sheets in regions 1-4
manifested a Cl
-positive Isc of
~32 µA/cm2 and average Gt of 9.4 ± 1 mS/cm2 under baseline conditions (n = 12 animals). In nonhyperproliferative regions of the Citrobacter
model (regions 1 and 2), these values remained
similar (Table 1). In partially hyperproliferative region 3,
baseline value of Isc was 31.7 µA/cm2
and Gt was 9.5 ± 1 mS/cm2. In the
fully hyperproliferative region 4 of the Citrobacter model, baseline value of Isc was 34.3 ± 3 µA/cm2 and Gt was 8.3 ± 1 mS/cm2, respectively (Table 1).

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Fig. 3.
Hyperplasia affects cAMP-dependent Cl secretion. Ion
transport measurements made across normal (open bars) and day
12 post-Citrobacter-infected hyperproliferative (hatched
bars) mouse colonic mucosal sheets. Bilateral addition of forskolin (10 µM) elicited changes in short-circuited Cl
secretory current (Isec) in all colonic segments,
which encompassed proximal (region 1) to distal (region
4) colonic boundaries. Mucosal hyperproliferation was associated
with dramatically enhanced short-circuit current
Isc responses to forskolin within distal colon
segments 3 and 4. All currents were inhibited by
serosal addition of furosemide (300 µM) or removal of bath
Cl . Values are means ± SD from 6 animals.
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These results clearly demonstrated in vivo that mucosal
hyperplasia within the distal colon was associated with enhanced
cAMP-dependent Cl
current per square centimeter of
luminal surface area. However, this analysis did not establish whether
increases in transmucosal CFTR-dependent Cl
secretory current reflects either an increase in the number of CFTR-containing colonocytes and/or an upregulation of CFTR
abundance/function within individual cells of the elongated crypt. To
begin to separate these cellular phenomena at the biochemical level,
quantitative estimates of cellular CFTR mRNA and protein abundance were
made in whole mucosa and isolated crypts.
Hyperproliferation Increases Mucosal Epithelial Cell CFTR mRNA and
Protein Expression
CFTR message and protein levels were determined in distal colonic
mucosa (regions 3 and 4) of normal and day 12 post-Citrobacter-injected mouse distal colon.
CFTR message levels.
Cellular CFTR poly(A)+ mRNA abundance relative to the
housekeeping gene GAPDH was observed to increase in both isolated
crypts (mean = 8.3 ± 0.2-fold) and whole mucosal tissue
(mean = 1.7 ± 0.2-fold) during colonic mucosal hyperproliferation
(Fig. 4A; n = 3 animals).
The intensity of the 6.5-kb CFTR band was normalized by stripping and
reprobing the blots for GAPDH. Normalization to the housekeeping mRNA
demonstrated that purified colonic crypts undergoing higher rates of
cell turnover exhibited higher average cellular CFTR message levels.

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Fig. 4.
Mucosal hyperplasia affects cystic fibrosis transmembrane conductance
regulator (CFTR) abundance. Cellular CFTR mRNA and protein measurements
were made in normal (N) and hyperproliferative (H) day 12 post-Citrobacter-infected animals. A, left:
Northern blot of poly(A)+ mRNA extracted from whole distal
colon (a) or isolated colonic crypts (b) probed with
CFTR (top) and glyceraldehyde 3-phosphate dehydrogenase
(bottom) oligonucleotide probes. A, right: normalized
relative cellular mRNA abundance levels for series of 3 blots from
whole distal colon (a) or isolated colonic crypts (b).
B, left: Western blot analysis of epithelial CFTR
probed with rabbit polyclonal anti-CFTR antisera (see
METHODS). Tissue extracts prepared from normal (N), day
12 (H1) and day 15 (H2)
post-Citrobacter-infected colon, or purified CFTR protein from
bovine trachea (C) are shown in a. Molar excess (+) or without
( ) immunizing peptide control for CFTR polyclonal antibody performed
in tracheal samples is shown in b. B, right: mean
relative protein abundance from 3 animals performed in duplicate.
Values are means ± SD. Mucosal hyperproliferation was associated with
increased cellular CFTR mRNA and protein expression.
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Cellular CFTR protein levels.
To test whether the changes in mRNA are also reflected at the protein
level, Western blot analysis was carried out in normal and day
12-15 post-Citrobacter-infected mice utilizing whole
distal colon tissue extracts from colonic segments 3 and
4. For this purpose, polyclonal anti-CFTR antibody made against
the COOH-terminal 13-amino acid cytoplasmic tail of purified bovine
CFTR with nearly complete homology to murine CFTR was used as a probe
(a kind gift from Dr. W. Dubinsky). When CFTR was immunoblotted for
these extracts, which were run with purified bovine CFTR as positive
control (Fig. 4B), an increase in cellular CFTR protein
expression normalized to
-actin was recorded (2.4 ± 0.2-fold
compared with normally proliferating mucosa). Antibody specificity was
demonstrated with molar excess of antigenic peptide from which the
antibody was raised (Fig. 4B, left, b; bovine
tracheal extract run on a separate gel). Because the rabbit
polyclonal antibody failed to detect a broad band of fully glycosylated
CFTR protein in any sample (Fig. 4B, left), this finding was
independently confirmed by Western blotting with TAM18 murine
anti-human whole molecule CFTR monoclonal antibody (Fig.
5).

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Fig. 5.
Western blot analysis of epithelial CFTR probed with TAM18 murine
anti-human CFTR monoclonal antibody detected with horseradish
peroxidase-conjugated goat anti-mouse IgM secondary antibody (see
METHODS). Tissue extracts were prepared from normal (N) and
transmissible murine colonic hyperplasia (TMCH) mouse distal colon (H).
A broad band of immunoreactive protein of ~190 kDa was detected in
both samples (C). Under hyperproliferatory conditions, this band was
much more intense (same amount of protein was loaded onto gel) and was
accompanied by less intense bands at ~140 (A) and ~160 (B) kDa. No
immunoreactive bands of molecular mass corresponding to endogenous IgM
heavy or light chains (55 and 30 kDa, respectively) were detected in
these samples.
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When utilizing the murine-derived IgM anti-human CFTR monoclonal
antibody, three immunoreactive bands were seen (Fig. 5). Band
C, corresponding to fully glycosylated forms of the protein, was
present in both normal and hyperproliferative crypts. Core glycosylated
protein (band A) and partially glycosylated protein (band
B) (20) were not clearly evident in normal crypt extracts but were
present in hyperproliferating crypts, although the latter band was
fainter. These differences in expression may simply reflect the higher
levels of fully glycosylated CFTR protein in both samples. Mucosal
crypt hyperproliferation was therefore associated with increases in the
cellular abundance of mature CFTR protein, which did not match
elevations in steady-state CFTR poly(A)+ mRNA levels.
The horseradish peroxidase-conjugated goat anti-mouse IgM secondary
antibody failed to detect endogenous levels of tissue-specific IgM (no
heavy- or light-chain IgM bands were seen on the CFTR Western blot;
Fig. 5). The murine anti-CFTR monoclonal antibody was used extensively
by us in the following studies for immunolocating CFTR within the mouse
crypt epithelium.
Increases in both CFTR-expressing cell number and subcellular CFTR
content occur in hyperproliferating crypts.
Although both molecular and biochemical analysis revealed that
average values of cellular CFTR expression increase in
hyperproliferating crypts, they do not directly address whether this
epithelial cell-specific induction of anion channel protein represents
either overexpression within specific regions of the crypt normally
expressing CFTR or new CFTR expression in crypt regions normally devoid
of or expressing low levels of this protein. To address this concern, we performed immunofluorescence localization studies in formalin- and
methanol-fixed crypts (see METHODS) isolated from the
distal colon of both normal and TMCH mice (Figs. 6-8 and
10-12).
CFTR immunoreactive protein was initially detected in isolated crypts
from day 12 post-Citrobacter-infected mouse distal
colon using the TAM18 anti-CFTR monoclonal antibody. Images were
collected with the CLSM (Fig. 6).

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Fig. 6.
Confocal laser scanning microscope (CLSM) images made in TMCH distal
colonic crypts at high spatial resolution (×400). Single 0.4-µm
midcrypt z-axis planes of cellular CFTR immunoreactive apical
pole labeling and accumulation of CFTR within subcellular apically
oriented perinuclear compartments recorded with TAM18 monoclonal
antibody are shown in A. Similarly processed crypts lacking
primary antibody displaying low background levels of immunostaining are
shown in B. Crypts in which anti-CFTR primary antibody was
replaced with anti-CD15 pan-lymphocyte-specific antibody exhibited
nonspecific staining (C). Staining obtained with goat
anti-mouse pan-IgM primary antibody detecting endogenous levels of
immunoglobulin within cellular basolateral pole (n = 18 crypt
preparations from 10 TMCH mice) are shown in D. Images shown
here and in Fig. 7 were collected using same image capture gains and
were not differentially enhanced.
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Figure 6A is a representative example of CFTR immunostaining in
a 0.4-µm (midcrypt) z-axis fluorescent light section obtained from segment 4 of the day 12 TMCH mouse colon. A
similar staining pattern has been reproduced in 18 separate animals at
this time point. CFTR immunoreactivity accumulated at or below the
cellular apical membrane (defined by us as the cellular apical pole)
and was extended throughout the longitudinal cellular axis of the crypt
(base-to-neck region). Staining within the cellular subapical pole also
extended internally toward the apical-lateral junction, more clearly
seen at higher spatial resolution as a subapical circumferential net
(Fig. 10B). CFTR was also found to accumulate in perinuclear
but apically oriented intracellular compartments throughout individual
cells within the crypt. Only background levels of CFTR basolateral
signal were detected in hyperproliferative crypts (Fig. 6A).
Simultaneously processed crypts from the same animal but controlled by
omission of the anti-CFTR primary antibody from the staining protocol
gave barely detectable diffuse levels of nonspecific immunostaining
(Fig. 6B), whereas replacement of the anti-CFTR primary
antibody with a matched anti-CD15 panlymphocyte-specific IgM monoclonal
primary antibody revealed that cellular CFTR immunostaining was
specific (Fig. 6C). Confirming the antigenic specificity of the
secondary antibody, which in this instance is being used to detect a
murine primary antibody in murine tissue, we also immunolocated native
IgM in the same crypt samples using a separate goat anti-mouse IgM
primary polyclonal antibody and FITC-conjugated rabbit anti-goat secondary antibody pair (Fig. 6D). (Note that the goat
anti-mouse IgM isoform-specific secondary antibody used for these
studies, already preabsorbed against human IgA and IgG but not their
murine counterparts, did not detect significant levels of these
endogenous immunoglobulins.) Endogenous levels of IgM
were found to be predominantly basolateral and mainly within the upper
reaches of the longitudinal crypt axis (crypt neck region). Clearly,
high cellular levels of CFTR were detected throughout the crypt axis of
hyperproliferating crypts, and CFTR immunostaining at the cellular
apical pole in hyperproliferating crypts was immunospecific.
For comparison, CFTR was also immunolocalized in simultaneously
processed normal crypts (Fig. 7).
Immunofluorescent light was collected from these crypts using the same
signal gains applied in Fig. 6. No postimage capture contrast
enhancement was performed. Because of the linear nature of the digital
CLSM detection system (captured 16-bit images contained 65,536 grey
levels), images in Figs. 6 and 7 were directly comparable. The
fluorescent light CFTR immunostaining pattern within the midcrypt (0.4 µm) z-axis from matched (segment 4) normal mouse
colon exhibited a very different staining pattern (Fig. 7A).
Although apical pole immunoreactivity was recorded in cells within the
lower (basal) regions of the longitudinal crypt axis (Fig. 7A),
overall levels were quantitatively much lower. The mean digital signal
collected from a 10-µm2 sample window was 23 ± 4% of
that recorded in protocol-matched hyperproliferating crypts and was
significantly less (P < 0.001; n = 16 individual
observations). However, unlike hyperproliferating crypts,
immunoreactivity was also recorded at or below the basal plasma
membrane in all cells within this structure. This staining pattern was
most prominent in the crypt neck region, where it extended into the
cellular basolateral membrane. Sample and protocol-matched control
crypts in which the anti-CFTR primary antibody was omitted (Fig.
7B) or replaced with a CD15 panlymphocyte-specific IgM
monoclonal antibody (Fig. 7C) failed to reproduce the apical or
basolateral staining pattern. The goat anti-murine IgM control
performed at the same time in the same crypt samples revealed that
endogenous IgM levels were higher when compared with hyperproliferating
crypts (Fig. 7D), with immunoreactivity being localized almost
exclusively to the cellular basal plasma membrane. Thus cells within
the basal region of normal crypts exhibited weak apical pole CFTR
staining and, additionally, a distinct basal cellular pole staining
pattern that was most prominent in cells within the crypt neck region. This result has been replicated in normal distal colonic crypts from
six animals.

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Fig. 7.
CLSM images made in age-matched distal colonic crypts from normal mice
at high spatial resolution (×400). Single 0.4-µm midcrypt
z-axis planes of TAM18 antibody-dependent CFTR
immunoreactive staining within apical subcellular pole of crypt basal
region cells and subcellular basal pole staining in crypt neck region
cells are shown in A. Similarly processed crypts lacking
primary antibody displayed low background levels (B). Crypts in
which anti-CFTR primary antibody was replaced with CD15 pan
anti-lymphocyte specific antibody exhibited nonspecific staining
(C). Staining obtained with goat anti-mouse pan IgM primary
antibody detecting endogenous levels of this immunoglobulin within
cellular basolateral pole are shown in D. Higher endogenous
levels of this immunoglobulin were detected throughout crypt axis
(n = 6 crypt preparations from 6 normal mice). Images were
collected using same image capture setting as Fig. 6 and were not
differentially enhanced.
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Although the appearance of CFTR immunoreactivity within the apical
cellular pole confirmed the observations made in cultured colonocytes
in vitro (14), our finding of a basolateral signal was unexpected. One
of the problems of detecting CFTR in native gastrointestinal epithelia
has been the question of specificity with regards to both primary and
secondary antibodies. As shown in Figs. 6D and 7D, the
basal plasma membrane region of mature epithelial crypt neck cells in
particular is a site at which both IgM (shown) and IgA (not shown)
accumulate. These molecules, complexed with secretory component, then
translocate across the epithelium, where they act as the primary host
defense mechanism (5). Even though no immunoreactive endogenous IgM
protein was detected in the apical plasma membrane in any instance
(n = 47 crypt preparations), and the anti-IgM isotype specific
secondary antibody did not detect significant levels of endogenous IgM
(see Fig. 5), we measured total cellular levels of both IgM and IgA in
isolated crypts from both sources (Fig. 8).

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Fig. 8.
Western blot of endogenous IgA and IgM levels in normal (N) and TMCH
(H) crypt extracts. In both instances, crypt hyperproliferation caused
a slight reduction in total amount of detectable protein. hc,
Immunoglobulin heavy chain; lc, immunoglobulin light chain.
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We found that both IgM and IgA levels were slightly reduced in
hyperproliferating distal colonic crypt samples, a finding that
corroborates earlier studies demonstrating that a reduction in both
secretory component and immunoglobulin immunoreactivity closely
parallels loss of differentiation within crypts in both benign and
malignant neoplastic disorders (10). However, the magnitude of this
change is unlikely to account for the normal crypt cellular basolateral
signal because the present studies have ruled out
nonspecificity as a problem for the secondary antibody and,
quantitatively, the increase in cellular basolateral signal is much
larger than any change in IgA/IgM immunoreactivity recorded by either
immunohistochemistry (Fig. 6D vs. Fig. 7D) or Western blotting (Fig. 8). We therefore cannot exclude the possibility that
this signal is either real (related to CFTR antigenicity) or represents
a protocol artifact that is selectively recorded in normal, but not
hyperproliferative, crypts. Because this antibody failed to reproduce
the basolateral staining pattern in human colonic cell lines (HT-29
Cl.19A, data not shown) we believe this staining to be nonspecific.
When the base region of normal crypts was imaged at even higher
magnification (×800), fine subcellular detail of the anti-CFTR immunoreactive staining pattern was recorded (Fig.
9). CFTR accumulated in the subapical pole
of colonocytes in punctate vesicular structures, and occasionally CFTR
immunoreactivity extended throughout the cell (Fig. 9B).
Vertically reconstructed sections at three locations separated by 50 µm along the longitudinal (x-y) crypt axis (Fig. 9C)
clearly showed that CFTR immunoreactivity was concentrated at the
apical pole in cells within the basal crypt region (Fig. 9C,
iii). In cells nearer the midcrypt region, a more intense perinuclear intracellularly localized CFTR staining pattern existed, with no staining being seen in either the nuclear (Fig. 9, B
and C) or cellular apical pole (Fig. 9C, i and
ii).

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Fig. 9.
CLSM high-power (×800) image of an isolated crypt from normal
mouse distal colon. Bright field (A) and CFTR immunoreactive
(B) signals from a single 0.4-µm midcrypt z-axis
fluorescence light plane detected with TAM18 monoclonal antibody. At
this resolution, punctate vesicular staining within cellular apical
pole was seen in crypt base colonocytes, with occasional cells
exhibiting extensive cytoplasmic staining (*). C:
cross-sectional reconstructions of 100 individual x-z (75 µm × 0.4 µm) axial planes at 3 50-µm intervals between mid-
(C, i) and basal (C, iii) crypt axes
showing immunoreactive CFTR signal within cellular subapical pole of
crypt region colonocytes. Nuclear region, devoid of immunoreactivity,
is marked (arrow in B and C).
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Midcrypt z-axis planes from hyperproliferating crypts revealed
even more detail to the cellular and subcellular CFTR staining profile
(Fig. 10). Because the tubular crypts
adhere to the coverslip at different points along their length,
undulation of the crypt created, at this optical resolution, areas
within the midaxis plane where individual cells were bisected both
across their apical plasma membrane and, in some cases, just below this
structure. A subapical circumferential staining pattern for CFTR was
seen in these later instances (Fig. 10B), coinciding with the
cellular plane of the zonula adherens. Intense apical pole staining
within cells of the crypt base (Fig. 10B, iv and v) was
replaced by less intense but still present apical pole staining in
crypt neck cells (Fig. 10B, ii). In contrast, apically
oriented perinuclear staining increased along the longitudinal
x-y crypt axis (base to neck). This gradient was much more
intense than that recorded in normal crypts (Fig. 9B). The
accumulation of CFTR immunoreactivity at this additional subcellular
site was also registered in reconstructed x-z-axis planes shown
at 50-µm intervals along the longitudinal (x-y) crypt axis
(Fig. 10C, i-v). Again, no immunoreactive staining was recorded over individual cell nuclei that form a circular band and
were used for orientation purposes (Fig. 10C,
ii).

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Fig. 10.
CLSM high-power (×800) image of an isolated crypt from TMCH mouse
distal colon. Bright field (A) and CFTR immunoreactive
(B) signals from a single 0.4-µm midcrypt z-axis
fluorescence light plane detected with TAM18 monoclonal antibody. At
this resolution, cellular gradient (crypt base-to-neck region) for CFTR
accumulation within subcellular apically oriented perinuclear
compartments is seen. Undulation of crypt reveals that CFTR staining
below apical plasma membrane is registered as a circumferential band,
closely resembling physical location and structure of cellular zonula
adherens (marked with circle). C: cross-sectional
reconstructions of 100 individual x-z (75 µm × 0.4 µm) axial planes at 5 50-µm intervals along neck (C,
i) and base (C, iii) crypt axis revealed that
pronounced cellular apical pole labeling within cells of crypt base was
replaced with more intracellularly located protein in neck colonocytes.
Nuclear region, devoid of immunoreactivity, is marked (arrow in
B and C).
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Further investigation into the appearance of subcellular CFTR
immunoreactivity revealed that elongated crypts accumulated more signal
within the apically oriented perinuclear region than their shorter
counterparts (Fig. 11). CFTR
immunoreactivity was nearly exclusively localized to the cellular
apical pole throughout the longitudinal crypt axis of short crypts
(<200 µm in length), which on average made up only a small fraction
(<10%) of the total number harvested from day 12 post-Citrobacter-infected mice (n = 48 animals; Fig.
11A). In the major portion of crypts isolated at this time
point, which range in length between 400-500 µm (68% of all
crypts), both base-to-neck perinuclear and neck-to-base cellular apical
pole labeling coexisted (Fig. 11B). In longer crypts, characterized by lengths >500 µm (~18% of population),
subcellular apically oriented perinuclear labeling predominated over
apical pole staining (Fig. 11C). Thus the subcellular CFTR
staining pattern in hyperproliferating crypts appeared to be modulated
by crypt length and, by inference, the timing of hyperproliferatory
change within this structure.

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Fig. 11.
Composite of single CLSM 0.4-µm midcrypt z-axis fluorescence
light planes taken at ×400 magnification showing TAM18 monoclonal
antibody-specific CFTR immunostaining pattern in 3 crypts of different
lengths from same TMCH mouse distal colon sample. CFTR was found
predominantly within cellular apical pole of short crypts (<200 µm;
A), whereas it accumulated within apically oriented perinuclear
locations in correspondingly longer crypts (B and C).
Subcellular location of CFTR appeared to be dependent on either time of
onset or duration of cellular hyperproliferative signal.
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Lastly, the murine monoclonal anti-human CFTR antibody was used
extensively for these studies because it was much more efficient at
discriminating CFTR at both the cellular apical plasma membrane and
within subcellular structures (staining was ~8-fold brighter than
that obtained with the rabbit anti-bovine CFTR polyclonal used for
Western blotting in Fig. 4). When normal and day 12 post-Citrobacter-infected hyperproliferating crypts were
simultaneously probed with the peptide-based anti-CFTR polyclonal
antibody and FITC-conjugated goat anti-rabbit secondary pair, the
following signals were detected (Fig.
12). Immunofluorescent light was
collected at matched signal gains. Apical cellular pole CFTR staining
was recorded in cells within normal crypt base (Fig. 12A).
Clear increases in apical plasma membrane and cellular CFTR
immunoreactivity were recorded in hyperproliferating crypts, with
staining extending throughout the crypt axis in a pattern very similar
to that recorded with the TAM18 anti-CFTR monoclonal antibody (Figs. 7,
10, and 11). Staining was, however, less intense (Fig. 12B).
Similar results have been previously reported in abstract form by us
(18). This independent finding with a different peptide-based
polyclonal anti-CFTR antibody clearly confirmed both the specificity of
the cellular apical pole CFTR immunoreactive signal and the fact that
both the number of cells expressing this anion channel as well as the
overall cellular level of CFTR increased during crypt
hyperproliferation. In no instance was a basolateral signal recorded in
either sample with this latter antibody (crypts isolated from 16 normal
and 8 day 12 post-Citrobacter-infected animals). Thus,
although in this instance the lower signal-to-noise ratio afforded by
the polyclonal anti-CFTR antibody may have possibly masked a
basolateral staining pattern, it is more likely that cellular basal
pole CFTR immunoreactivity detected in normal crypts with the TAM18
monoclonal antibody is nonspecific.

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Fig. 12.
CFTR immunofluorescence staining pattern detected in age-matched
normally proliferating (A) and day 12 post-Citrobacter infected hyperproliferating (B) crypts
probed with rabbit anti-bovine CFTR peptide-based polyclonal antibody.
Both images were collected at ×400 using same capture gains and
were not differentially enhanced. Confirming results obtained with
TAM18 anti-CFTR monoclonal antibody, cellular apical pole CFTR
immunoreactivity greatly increased during crypt hyperproliferation
(n = 22 animals; see RESULTS).
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 |
DISCUSSION |
In Vivo Effects of Hyperproliferation on CFTR Abundance and Function
Previously, we and others (Ref. 13, reviewed in Refs. 7
and 12) have demonstrated that CFTR mRNA and protein expression in
colonic cell lines does not correlate with cAMP-dependent
Cl
anion transport. Both CFTR message and protein
levels remain unaltered in transformed colonocytes regardless of
whether they were proliferating or undergoing contact-induced growth
cessation. CFTR-dependent anion transport was, however, dependent on
differentiation-specific changes in cytoplasmic polarization and apical
plasma membrane CFTR targeting (13, 14). To address whether cellular
proliferatory status likewise failed to affect native
cell CFTR expression while inhibiting in vivo CFTR-dependent
Cl
secretion, we utilized the TMCH model of mucosal
hyperplasia (Fig. 1). In this model increases in proliferating
colonocyte number were seen: elongated crypts contained a smaller
percentage of mature goblet cells and exhibited packing of
nonvacuolated cells within the middle to lower crypt regions (Fig. 1),
and BrdU labeling was found throughout the crypt axis. The fact that
apoptosis was unchanged explained our reported eightfold increase in
proliferatory index (Fig. 2). TMCH-dependent increases in transmucosal
CFTR-dependent Cl
current generation (Fig. 3)
established that proliferatory conditions within the epithelium
promoted rather than inhibited secretory function at the tissue level.
Native colonocytes even at the base of the crypt possess tight
junctions, are cytoarchitecturally polarized (8), and are thus, by in
vitro standards, differentiated. Thus there were clearly important
differences between transformed cell lines and native colonocytes.
To begin to address the nature of these differences, we tested the
hypothesis that the enhanced Cl
secretory response
of TMCH mucosa was due to either an increase in CFTR-containing cell
number within the crypt unit and/or an increase in CFTR anion channel
expression in individual cells within the crypt. In fact, we found that
normalized cellular levels of both CFTR mRNA and protein were higher in
hyperproliferating crypt cells (Fig. 4). However, whereas cellular
poly(A)+ mRNA expression increased 8-fold, only a 2.4-fold
increase in cellular protein was recorded. Colonocytes are estimated to
take 16-18 h to traverse ~200-µm-long normal crypts (see
reviews in Refs. 12 and 20), whereas CFTR protein turnover rate
(production and degradation) has been estimated in vitro to be on the
order of 7-12 h. Thus we concluded that either colonocytes fail to
remain within the crypt unit long enough to attain maximal levels of CFTR protein or that elevated endogenous poly(A)+ CFTR
message was inefficiently translated. Given that CFTR transcript levels
are not characterized as being abundant (6, 11, 15), it seemed unlikely
that the cellular biosynthetic capacity for CFTR had been reached.
Rather, our studies suggested that posttranslational modes of CFTR
regulation were present within native colonocytes that protect the cell
from the pathophysiological consequences of excessive anion channel expression.
A corollary of our above hypothesis was that CFTR anion channel protein
expression was predicted to extend into the neck and surface regions of
hyperproliferating crypts. [This was theorized on the basis of
the short transit time for cell movement along the crypt axis, the long
half-life of cellular CFTR protein turnover, and the fact that normally
only a small proportion of cells within the crypt express detectable
CFTR mRNA levels (6).] To test this hypothesis, we quantitatively
measured CFTR immunoreactivity in paired crypt preparations from normal
and TMCH mice using two methods of fluorescent light microscopy. We
found that CFTR expression was indeed extended into neck regions of the
hyperproliferating crypt (Figs. 6A, 10B, 11, and 12).
Furthermore, we found that total subcellular levels of immunoreactive
CFTR protein were higher in hyperproliferating crypt colonocytes than
their normal crypt counterparts (Fig. 6A vs. Fig. 7A;
see RESULTS). Thus elevated native mucosal proliferation
promoted increased cellular CFTR protein levels both within areas in
which CFTR was normally detected and in regions in which CFTR was
undetectable under normal conditions. Hyperproliferating colonocytes,
although structurally more polarized than their cell line counterparts,
therefore differ in an important respect: their CFTR message levels are
dramatically altered by their proliferatory status (13).
The second major finding of this study was that CFTR accumulated in
apically oriented perinuclear structures in hyperproliferating crypts
to a much larger extent (Fig. 6A and Fig. 10, B and
C) than that observed in normal crypts (Fig. 7A and
Fig. 9, B and C). We found that accumulation within
this structure was dependent on crypt length (Fig. 11), suggesting that
either the onset or duration of the hyperproliferatory signal was
important. The fact that short hyperproliferating crypts exhibited
nearly exclusive apical pole CFTR labeling (Fig. 11A), whereas
elongated hyperproliferating crypts exhibited mainly perinuclear
labeling (Fig. 11C), allowed us to theorize where in the cell
this pool of CFTR had originated from. The glycosylation pattern of
CFTR Western blotted with TAM18 antibody demonstrated that mature
(post-Golgi-processed) protein was overproduced by hyperproliferating
colonocytes (Fig. 5). We suggest that anion channel retrieval from the
apical pole or late stages of the biosynthetic pathway, rather than
inhibition of nascent channel movement from within early (endoplasmic
reticulum) compartments, may explain this phenomenon.
Subcellular biochemical and structural studies are currently underway
to test the apical plasma membrane CFTR retrieval hypothesis, which we
believe may serve as an important physiological defense mechanism
against cellular CFTR overexpression in vivo. This could explain why
CFTR-mediated current generation across TMCH distal colon did not
greatly exceed the theorized twofold increase in mucosal surface area
predicted by a twofold elongation in crypt length (Fig. 1). These
findings highlight an important new aspect of CFTR regulation
in vivo.
 |
ACKNOWLEDGEMENTS |
This work was supported by funds from the Cystic Fibrosis
Foundation and the American Institute for Cancer Research.
 |
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: A. P. Morris,
Dept. of Internal Medicine, Division of Gastroenterology, Hepatology
and Nutrition, The Univ. of Texas Health Science Center at Houston,
Medical School, Houston, Texas, 77030 (E-mail:
amorris{at}girch1.med.uth.tmc.edu).
Received 3 March 1999; accepted in final form 15 December 1999.
 |
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