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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In the companion
article (Umar S, Scott J, Sellin JH, Dubinsky WP, and Morris AP, Am
J Physiol Gastrointest Liver Physiol 278: 753-764, 2000),
we have shown that transmissible murine colonic hyperplasia (TMCH)
increased cellular cystic fibrosis transmembrane conductance regulator
(CFTR) mRNA and protein expression, relocalized CFTR within
colonocytes, and enhanced mucosal cAMP-dependent Cl
secretion. We show here that these changes were dependent on elevated
cellular levels of membrane-bound Ca2+- and
diacylglycerol-sensitive protein kinase C (PKC) activity (12-fold), induced by selective (3- to 4-fold) rises in conventional PKC (cPKC) isoform expression and membrane translocation. Three cPKC
isoforms were detected in isolated crypts:
,
1, and
2. cPKC-
1 rises preceded and those of cPKC-
and cPKC-
2 paralleled cellular hyperproliferation and its effects on CFTR expression and
cAMP-dependent Cl
current secretion. Only cPKC-
1
and cPKC-
2 were membrane translocated during TMCH. Furthermore, only
cPKC-
1 trafficked to the nucleus, whereas cPKC-
2 remained
partitioned among cytosolic, membrane, and cytoskeletal subcellular
fractions. Modest increases in novel PKC-
(nPKC-
) expression and
subcellular membrane partitioning were recorded during TMCH, but no
changes were seen for PKC-
or -
. No nPKC isoform nuclear
partitioning was detected. The orally bioactive cPKC inhibitor
Ro-32-0432 reversed both TMCH and elevated cellular CFTR mRNA
levels, whereas a pharmacologically inert analog
(Ro-31-6045) failed to inhibit either response. On the basis of these facts, we present a new hypothesis whereby PKC-dependent cellular proliferation promotes endogenous cellular CFTR
levels. PKC-
1 was identified as a candidate regulatory PKC isoform.
protein kinase C; cystic fibrosis transmembrane conductance regulator; anion transport; regulation; mouse
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INTRODUCTION |
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PROTEIN KINASE C (PKC) consists of a family of
serine/threonine kinases shown to be involved in the regulation of many
aspects of cell growth, differentiation, and function (6). In the
context of epithelial Cl secretion, PKC has been
implicated in the regulation of both apical and basolateral plasma
membrane ion transport events (9, 15, 37). The cell biology of
epithelial cAMP-stimulated anion transport in vitro is dominated by
reports demonstrating a negative role of phorbol ester-sensitive PKC
signaling in cystic fibrosis transmembrane conductance regulator (CFTR)
expression. Chronic PKC stimulation by phorbol ester treatment is
believed to lead to downregulation of CFTR mRNA and possibly protein
levels (1, 12, 36, 38). However, phorbol esters can also mediate
positive effects on the cellular function of CFTR. They have been shown to potentiate cAMP-responsive Cl
current generation
in epithelial cells through phosphorylation-dependent effects on
channel gating (4, 19 ). They are also predicted to enhance the apical
plasma membrane accumulation of this anion channel through stimulatory
effects within the cellular biosynthetic pathway (25).
However, a recognized caveat of phorbol ester use both in vitro and in vivo is their ability to promote both negative and positive effects on cellular signaling, depending on their time of application and dose. The broad stimulatory effects of these artificial agents in cells may also be accompanied by nonspecific actions on multiple cellular targets. Given that we had found that proliferation in vivo promoted native colonocyte CFTR mRNA and protein levels (see companion paper, Ref. 38a), we measured phorbol ester-sensitive conventional PKC (cPKC) and novel PKC (nPKC) activity during transmissible murine colonic hyperplasia (TMCH) to determine whether individual or multiple isoforms were downregulated during periods of enhanced CFTR expression in vivo. Our studies found that native tissue PKC activation does not mimic the in vitro effects of phorbol esters. cPKC activity increased in the TMCH model. Furthermore, pharmacological inhibition of cPKC activity in vivo prevented both TMCH and enhanced cellular CFTR expression levels. Three participating cPKC isoforms were identified.
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METHODS |
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Reagents.
Polyclonal rabbit anti-PKC-, PKC-
1 anti-peptide, and monoclonal
anti-PKC-
antibodies were purchased from Sigma Immunochemicals (St.
Louis, MO). PKC-
2, -
, -
, and -
anti-peptide antibodies were
procured from Santa Cruz Biotechnology (Santa Cruz, CA). Ro-32-0432 and Ro-31-6045 were purchased from Alexis
Biochemicals (San Diego, CA). Succinylated gelatin carrier
(Gelofusine) was a kind gift from B. Braun Medical
(Aylesbury, UK).
PKC activity assay.
Conventional Ca2+- and diacylglycerol-sensitive PKC (cPKC)
activity was measured in cytosolic and membrane fractions of isolated crypt, crypt denuded colon, and whole proximal and distal colon samples
from normal and day 12 post-Citrobacter-infected
animals using a PKC assay system (GIBCO BRL, Grand Island, NY).
Subcellular fractions were prepared by homogenization of both isolated
crypts and whole distal colon tissue in 1.5 ml of ice-cold
homogenization buffer (50 mM Tris · HCl, pH 7.5, containing 5 mM EDTA, 10 mM EGTA, 0.3% mercaptoethanol, 10 mM
benzamidine, 50 µg/ml phenylmethylsulfonyl fluoride, and 50 µg/ml
leupeptin) using a Tekmar Tissuemizer (Tekmar, Cincinnati, OH) rotating
at 20,000 rpm for five 40-s periods. Cytosolic and membrane fractions
were separated by sedimentation for 30 min at 100,000 g. The
resulting pellet was then detergent extracted in buffer (1% Triton
X-100, 10 mM Tris · HCl, pH 7.5, 140 mM NaCl, 25 mM
KCl, 5 mM MgCl2, 2 mM EDTA, and 2 mM EGTA) containing
protease inhibitors, and the solubilized membrane fraction was
collected by centrifugation. Protein content was determined using the
Bio-Rad DC protein assay kit. PKC activity was quantified by the
transfer of the terminal phosphate of
[-32P]ATP (Amersham, Arlington Heights, IL)
to myelin basic protein synthetic peptide (41). cPKC specificity was
determined by using a cPKC pseudosubstrate inhibitor peptide
[PKC-(19-36)] provided by the manufacturer.
Incorporated radioactivity was determined by scintillation counting,
and activities were expressed as picomoles of phosphate transferred per
minute per milligram of protein.
Tissue preparation for Western blot analysis. Male Swiss Webster mice (15-20 g; Harlan Sprague Dawley, Houston, TX) were killed by cervical dislocation 0, 1, 3, 6, 9, 12, and 15 days after Citrobacter inoculation. The entire distal colon was removed, flushed with ice-cold PBS, and cut open longitudinally. Crypts were then isolated by attachment of the colonic sheet to a paddle followed by immersion 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) maintained at 37°C and 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. The remaining crypt-denuded colon was then saved for biochemical analysis of PKC activity. Control crypts were similarly isolated from the entire (~3 cm long) proximal colon of TMCH animals for cPKC activity measurements.
Crude cellular homogenates were prepared from isolated crypts taken from three normal and Citrobacter-infected animals per experimental observation by homogenization in detergent containing buffer (in mM: 50 Tris · HCl, 250 sucrose, 2 EDTA, 1 EGTA, pH 7.5, and 10 2-mercaptoethanol, with 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. Subcellular membrane and cytosolic fractions were prepared from similar crypts by omitting detergent and by centrifugation at 100,000 g. The resulting pellet was then detergent extracted (1% Triton X-100, 10 mM Tris · HCl, pH 7.5, 140 mM NaCl, 25 mM KCl, 5 mM MgCl2, 2 mM EDTA, 2 mM EGTA, and protease inhibitors), and the solubilized membrane fraction was collected by centrifugation. The detergent-insoluble pellet was solubilized in RIPA buffer (PBS, 1% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate) for estimates of cytoskeletal-associated protein. Nuclear extracts were prepared from freshly isolated mucosal tissue by the method of Zhang and colleagues (42). Protein concentration was measured in cytosolic, membrane, cytoskeletal, and nuclear fractions before electrophoresis. Mouse brain homogenates acted as positive controls for PKC isoform immunoblotting assays. Total cell extracts or subcellular fractions (30 µg protein/lane) were 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 PKC isoform/CFTR antibodies (0.5-1.0 µg/ml in TBS containing 0.1% Tween 20; Sigma). After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or rat anti-mouse IgG and developed using the ECL detection system (Amersham) according to the manufacturer's instructions. To assay for purity of cytosolic, membrane, cytoskeletal, and nuclear fractions, the following non-PKC-related marker proteins were detected by Western blotting (data not shown): 1) Lamin B was used to assay nuclear purification. Lamin B does not partition into other cellular compartments, and, by this test, both membrane and cytosol were free of nuclear contamination. 2) The transmembrane protein E cadherin was used as a marker for the membranous fraction. E cadherin does not translocate into the nucleus and is not freely cytosolic. Both nuclear and cytosolic fractions were free of this marker, indicating negligible cytoplasmic contamination. 3) MonomericCellular mRNA extraction.
Total mRNA was isolated from whole distal colon of normal and
Citrobacter-infected mice using TRIzol reagent (GIBCO BRL)
according to the manufacturer's instructions. For Northern blot
analysis, each preparation (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 an
[-32P]dCTP-labeled probe encompassing the R
domain of CFTR (bases 1773-2654; 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. Both were confirmed by oligonucleotide sequencing before random
primed labeling.
Development of TMCH. TMCH was developed in male Swiss Webster mice by oral inoculation with 16-h culture of Citrobacter freundii (biotype 4280, ATCC). Age-matched control mice received sterile culture medium only. Additional methodological details are given in the companion article (38a).
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RESULTS |
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Analysis of PKC activity in subcellular fractions of
isolated crypts, crypt-denuded colon, and whole colon tissue.
Total conventional Ca2+- and
diacylglycerol-sensitive cPKC activity in cytosol and membrane
fractions prepared from purified distal colonic crypts, crypt denuded
distal colon, or whole distal colon tissue were measured before
(day 0), and during (day 12 post-Citrobacter
infection) the peak mucosal hyperproliferatory response (38a).
Enzymatic activity, recorded as phosphate incorporation into a
synthetic substrate peptide (see METHODS), was detected in
both fractions (Fig. 1A).
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Crypt hyperproliferation was associated with selective changes in
PKC isozyme abundance.
To date, 12 PKC isozymes (,
1,
2,
,
,
,
,
,
,
,
, and µ) with differing structure, substrate
specificity, cofactor requirements, tissue expression, and localization
have been identified. This diversity is believed to create the varied
consequences of PKC activation in the same cell and are the reason why
individual isozymes are thought to possess distinct and specialized
functions in cell signaling (6).
cPKC expression in isolated crypts from normal and TMCH mice.
Individual cPKC isozyme expression was measured in Triton
X-100-solubilized crypt extracts prepared from the distal colon of
animals at 0-15 days after Citrobacter infection by
Western blot analysis. As shown in Fig. 2A,
mean cellular PKC- expression increased 1.6-fold during crypt
hyperproliferation. The mean relative cellular optical density of
PKC-
1
increased even more dramatically (3.7-fold; n = 3 animals in duplicate). Changes in PKC-
1 expression were
significantly different by the third day after inoculation and
continued to be so for all subsequent days (Fig. 2B;
at
day 3 = 0.6 ± 0.06 and normal = 0.38 ± 0.1; P < 0.001, Student's t-test; n = 6). This increase
occurred before the onset of marked crypt cell hyperplasia at day
6 (2, 35). Large twofold increases in crypt-specific
were also observed (n = 3 animals in duplicate; Fig.
2C). However, like PKC-
, statistically significant changes
were not recorded until nine days after inoculation
(
at day 9. = 0.76 ± 0.07 and normal = 0.38 ± 0.01; P < 0.01;
at
day 9 = 1.2 ± 0.18 and normal = 0.73 ± 0.08; P < 0.001; n = 6). Thus, unlike PKC-
1, crypt-specific changes in
PKC-
2 and PKC-
cellular expression paralleled the mucosal
hyperproliferatory response. PKC-
was undetectable in isolated crypt
extracts. Antibody specificity was confirmed by using the relevant
antigenic peptide for competition at a 1:2 (wt/wt) peptide-to-antibody
ratio (data not shown).
-Actin housekeeping protein antisera were
used to normalize variations in protein loading.
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Crypt colonocyte hyperproliferation was associated with enhanced cytosol-to-membrane translocation of all cPKC isoforms. It is widely accepted that PKC exists in an inactive conformation in the cytosol and that activation of the enzyme results in its translocation from the cytosol to membranes (6). Thus an indication of the native activation status of PKC can be obtained by assessing its distribution among subcellular compartments of the cell.
Figure 3 shows a representative Western blot and corresponding mean densitometric ratio for the membrane-to-cytosolic partitioning (Rm:c) of all three cPKCs detected in isolated crypt cellular extracts from normal and hyperproliferative TMCH mucosa.
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nPKC isoform expression during TMCH.
The expression of individual nPKC isozymes was likewise measured in
Triton X-100-solubilized crypt extracts prepared from the distal colon
of normal and TMCH animals. A panel of isozyme-specific polyclonal antibodies were used as probes for Western blot analysis. The specificity of each antibody was determined by competitive blotting with corresponding immunizing peptides, and their relative abundance was compared with mouse brain nPKC homologs (normalized to
-actin to account for differences in gel loading). Three novel PKC
isoforms,
,
, and
, were expressed in isolated crypts (Fig. 4A).
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Crypt colonocyte hyperproliferation was associated with modest
changes in cytosol-to-membrane translocation of specific nPKC isoforms.
The distribution of crypt-expressed nPKC isozymes was assessed in
cytosol and membrane fractions from normal and day 12 post-Citrobacter-infected (TMCH) mice (Fig.
5). A representative Western blot (Fig.
5A) and corresponding mean densitometric Rm:c for
nPKC (Fig. 5B) demonstrated the following: PKC- did not
exhibit significant increases in membrane association during TMCH.
PKC-
Rm:c was 1.12 ± 0.4-fold of normal values.
However, cytosolic PKC-
levels (Fig. 5A), quantified by
reprobing the same blots with antisera against
-actin, exhibited an
18% (1.27 ± 0.2-fold) decrease during crypt hyperproliferation, but
low levels of expression of this protein made it difficult to determine
whether these changes were real. PKC-
expression changes in TMCH
crypts (Fig. 4B) were paralleled by a concomitant increase in
the membrane translocation. The Rm:c increased 2.16 ± 0.15-fold above normal values. This correlated with a 32% decease in
the relative cytosolic abundance of PKC-
during crypt
hyperproliferation (
normal colon = 0.48 ± 0.06 and day 12 post-Citrobacter infected colon = 0.29 ± 0.05). Thus crypt hyperproliferation was also correlated with both early changes in
colonocyte PKC-
expression and biochemical activation, as measured
by this isoform's translocation to membrane fractions. PKC-
detected in isolated crypts exhibited a modest but insignificant increase in membrane association during crypt proliferation. The Rm:c increased 1.3 ± 0.2-fold compared with control
values. However, cytosolic expression of this isoform was barely
detectable either in normal mice or during crypt hyperproliferation and
again were so low as to hinder accurate measurement.
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Isoform-specific nuclear partitioning occurs in hyperproliferating
crypts.
Our immunological studies identified four phorbol ester-sensitive PKC
isoforms with increased cellular expression and intracellular membrane
activation status during TMCH: cPKC-, cPKC-
1, cPKC-
2, and
nPKC-
. To determine whether these PKC isoforms accumulated in other
subcellular locations, purified isolated nuclei from normal and TMCH
colon were probed by Western blotting (Fig.
6). Low levels of nuclear PKC-
1
immunoreactivity were detected in normally proliferating crypts,
whereas hyperproliferating crypts exhibited dramatically increased
levels of this kinase (Fig. 6). In the nuclei from hyperproliferating
mucosa, PKC-
2 levels were too low to quantify effectively (Fig. 6).
Densitometric analysis revealed that the PKC-
1 nuclear signal (Fig.
6, top) was ~18.5 ± 4.5% of the total
immunoreactivity detected in the pooled Triton X-100-extractable
membrane and cytosolic fractions in TMCH crypts (n = 2 animals
in duplicate). Nuclear PKC-
2 levels accounted for <2% of this
fraction (Fig. 6, top). Nuclear PKC-
1 immunoreactivity was
competed by immunizing peptide (data not shown). Purity of the nuclear
fraction was estimated by the presence of nuclear lamin B (Fig. 6,
middle) and exclusion of monomeric
-actin immunoreactivity (Fig. 6, bottom). The latter was detected only in total
solubilized cell extract and in cytoplasmic cellular extracts but not
in purified nuclear extract. PKC-
, -
2, and -
were not detected
in normally proliferating crypt nuclei. Although we cannot rule out the
possibility that antibody affinity may account for the lack of nuclear
signal recorded by these isoforms, the results clearly demonstrated
that a significant proportion of PKC-
1 was translocated into the
nucleus of proliferating colonocytes.
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The PKC-2 homolog of PKC-
1 remained
predominantly cytoskeletal-associated in both normal and
hyperproliferative crypt mucosa.
Triton X-100-insoluble RIPA-solubilized extracts from normal and
day 12 post-Citrobacter-infected mouse
whole distal colon revealed significant PKC-
2 immunoreactivity (Fig.
7). In the cytoskeletal fraction of both
normal and hyperproliferating crypts, PKC-
2 holoenzyme (80 kDa)
accounted for <5% of immunodetectable densitometric signal (Fig. 7).
The majority (>95%) of PKC-
2 was detected at ~50 kDa (Fig. 7).
Both bands were competed away with the PKC-
2 COOH-terminal peptide
(amino acids 657-673; peptide-to-antibody ratio = 1:1 wt/wt)
against which the antibody was raised (Fig. 7, middle). The
50-kDa immunoselective band was occasionally detected in Triton X-100
solubilized fractions (3 out of 12 blots), where it comprised between 1 and 12% of the 80-kDa PKC-
2 signal (Fig. 7; n = 12), which
suggests it is a proteolytic product of the holoenzyme. When
densitometric analysis of the total cytoskeletal and membrane-bound
PKC-
2 immunoreactivity were compared, modest increases in Triton
X-100-extractable signal during TMCH (Fig. 7; see also Fig. 3) were
reflected by modest decreases in cytoskeletal signal (Fig. 7). This
movement may account in part for the modest increases in cellular
expression levels (Fig. 2) and cytosol-to-membrane partitioning (Fig.
3) seen for PKC-
2 during TMCH. No PKC-
1 immunoreactivity was
detected in the cytoskeletal fractions of either normal or hyperproliferating mucosa (Fig. 7, bottom). These findings were confirmed in four uninfected/infected animals in duplicate.
Cytoskeletal levels of nPKC-
were not quantified.
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Citrobacter-induced crypt extension and elevated
cellular CFTR mRNA levels were inhibited by Ro-32-0432
but not by its pharmacologically inactive analog
Ro-31-6045.
The possibility that nuclear translocated PKC-1 was responsible for
TMCH-dependent increases in cellular CFTR message levels was
investigated by dosing mice with the highly selective cPKC antagonist
bisindolylmaleimide XI · hydrochloride
(Ro-32-0432). Ro-32-0432 was given periorally one day
before Citrobacter inoculation, and booster doses were
administered daily over the following two weeks. Ro-32-0432 has
previously been shown to be an effective in vivo antiarthritic agent
capable of inhibiting phorbol ester-induced paw edema in rats
(ED50 = 11 mg/kg; Ref. 5) but had not before been used in mice. This agent inhibits endogenous cPKCs with 100- to
10,000-fold or more selectivity over a variety of other
serine/threonine kinases in vitro (inhibition
constant =10-30 nM). However, higher doses have been
found to be required in vivo because endogenous ATP competes with this
agent for the ATP hydrolysis site of PKC (5). Groups of five mice were
dosed at 10 mg/kg body wt with Ro-32-0432 plus carrier or with
carrier alone (4% succinylated gelatin; see Table
1).
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DISCUSSION |
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The cell biology of CFTR expression in vitro is dominated by reports investigating the negative role of phorbol ester-sensitive PKC signaling (reviewed in Ref. 26). We therefore examined PKC expression and function in the TMCH model in which cellular CFTR levels increased (38a).
Cellular roles of individual PKC- isoforms in
proliferation-dependent changes in CFTR expression.
Although PKC activation has been implicated in colonic epithelial cell
proliferation in response to various stimuli, very little information
regarding the role of individual PKC isoforms exists. Rat and human
colonic mucosa have been shown to express both mRNA and protein for six
PKC isoforms (
,
,
,
,
, and
) (13, 20). In the
present study we concentrated on phorbol ester-sensitive
conventional and novel PKCs in which a distinct pattern of
cPKC-
, -
1, -
2, -
, -
, and -
isozyme expression and activation status was recorded.
PKC-.
Although isolated crypt colonocytes increased their levels of cPKC-
during TMCH (Fig. 2), no significant changes in membrane translocation
were recorded (Fig. 3) and PKC-
was undetectable in nuclear extracts
from either control or hyperproliferating crypts (data not shown). Thus
we found no evidence that PKC-
acts as either a negative regulator
of cellular CFTR expression, as suggested by in vitro studies (12), or
as positive regulator of CFTR expression in the in vivo TMCH
gastrointestinal epithelium.
PKC-.
Various studies have shown that PKC-
can regulate epithelial cell
cytokinetics. The role of PKC-
1 has been demonstrated in colonic
tumor cell lines in which growth cessation correlated with a selective
3-fold decrease in PKC-
1 abundance, a 10-fold decrease in
membrane-bound cPKC activity, and loss of mitogen-kinase cell signaling
(22). PKC-
heterologous overexpression has been shown to cause
dedifferentiation, proliferation, and an enhanced growth of colonocytes
in athymic mice (34). Marked increases in both cPKC-
1 and -
2
abundance (Fig. 2) and membrane translocation (Fig. 3) indicated that
these isoforms contributed the most to the 12-fold increase in
particulate cPKC activity recorded in hyperproliferating crypts (Fig.
1). Temporal differences in their cellular expression and subcellular
organelle location allowed us to further distinguish roles for these
two isoforms.
PKC-1.
Control crypts normally exhibited low cytosolic and membrane levels of
PKC-
1 (Figs. 2 and 3). However, colonocyte levels of PKC-
1
increased at the onset of TMCH before both PKC-
, PKC-
2, and gross
changes in mucosal mass occurred. In hyperproliferative mucosa, ~20%
of PKC-
1 immunoreactivity was also relocalized into the nucleus
(Fig. 6), whereas <2% was detected in nuclear extracts from normal
crypts. These findings predict a role for PKC-
1 in early nuclear
events involved in crypt proliferation and accompanying increases in
colonocyte CFTR expression.
PKC-2.
Increased crypt levels of PKC-
2 followed six days later than
PKC-
1 in the TMCH model (Fig. 2), at a time when dramatic increases in PKC-
abundance and mucosal mass occurred. Hyperproliferating crypts at day 12 contained 2.3-fold higher levels of
membrane-bound PKC-
2 (Fig. 3). Additionally, this isoform was
partitioned into cytoskeletal fractions under both normal and
hyperproliferative conditions (Fig. 7), whereas vanishingly low levels
of nuclear immunoreactivity were detected in both normal and
hyperproliferating crypts (Fig. 6). Given these findings, PKC-
2
appears less likely than PKC-
1 to mediate early
proliferation-dependent nuclear events leading to enhanced cellular
CFTR mRNA expression.
PKC-.
Previous studies relating the expression and cellular roles of PKC-
have shown both positive and negative effects on cell division (17). In
general, expression is seen predominantly in postmitotic cells of the
upper crypt and surface mucosa (39). In our model, colonocytes
increased their levels of PKC-
slightly during TMCH (Fig. 4).
However, no significant changes in membrane translocation were recorded
during TMCH, and PKC-
was undetectable in the purified nuclear
extracts from either control or hyperproliferating mucosa (data not
shown). Thus PKC-
may not be critical to
hyperproliferation-dependent changes in cellular CFTR expression.
PKC-.
Although PKC-
is expressed at very low levels in all normal tissues
except for brain, it is expressed at high levels in several hemopoietic
cell lines and tumors (24). During periods of mucosal hyperproliferation, PKC-
expression in the crypt increased, with concomitant increase in membrane translocation and hence cellular activation status (see Figs. 4 and 5). However, despite these changes,
nuclear translocation of PKC-
was not observed. This was unexpected
because PKC-
has been established in other systems to possess
oncogenic potential and, furthermore, the overexpression of PKC-
had
been implicated in ras-mediated signal transduction during
neoplastic transformation of the colonic epithelium (31). However, TMCH
does not cytologically correspond to neoplasia or tumorigenic
transformation (3), and the lack of nuclear PKC-
partitioning may
reflect this fact. Therefore, despite being the PKC isoform with
best-described oncogenic potential, it is difficult to postulate a
direct role for PKC-
in the regulation of TMCH-dependent nuclear
CFTR gene transcription. However, studies in several nonepithelial cell
lines have shown that both mitogenic and differentiating factors (27)
may use PKC-
signaling at the cellular plasma membrane to
affect responses at the genome level (33, 40). Thus we
cannot exclude the possibility that PKC-
can activate protein kinase
cascades within the nucleus without being physically present there. In
vivo transgenic or epigenic approaches will be required to determine
whether this isoform actively participates in the CFTR expression response.
PKC-.
No evidence was found for the participation of this isoform in
proliferation-dependent changes in cellular CFTR expression during TMCH.
In vivo effects of PKC signaling on CFTR abundance differ
from those proposed in vitro.
cPKCs are identified as the major cellular receptor for active phorbol
esters (8). Phorbol esters have been shown to acutely increase
electrogenic Cl secretion in ex vivo colonic
mucosa (9, 15), predominantly through cPKC activation and
secondary PGE2 production (37), and to directly stimulate
CFTR current generation in isolated epithelial cells (4, 19).
Phorbol ester-dependent stimulation of cPKC activity in vivo
also induces enhanced proliferation (7, 11) within the same
crypt cell populations we have shown to overexpress CFTR protein
(38a). However, phorbol esters and calcium ionophores are also
known to downregulate cellular CFTR mRNA levels within 1-12 h
[by as much as 80% within 2 h (36), but found to downregulate
less by other groups (1, 38)] in vitro and, in a separate
corroborating in vitro study, ras-induced PKC-
activation has been reported to both decrease cellular CFTR mRNA levels
and inhibit cAMP-dependent Cl
secretion (12). How
then can the negative effects of phorbol esters on cellular CFTR
expression in vitro be reconciled with the positive effects of
cPKC and nPKC activation recorded in the TMCH model in vivo?
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ACKNOWLEDGEMENTS |
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This work was supported by funds from the Cystic Fibrosis Foundation, the American Institute for Cancer Research, and the Cancer Research Foundation of America.
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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, TX 77030 (E-mail: amorris{at}girch1.med.uth.tmc.edu).
Received 15 October 1999; accepted in final form 2 December 1999.
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