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secretion
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 |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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).| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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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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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Mucosal Hyperproliferation Affects cAMP-Mediated
Cl Secretion
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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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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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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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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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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.
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ACKNOWLEDGEMENTS |
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This work was supported by funds from the Cystic Fibrosis Foundation and the American Institute for Cancer Research.
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FOOTNOTES |
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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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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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) 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.
