Increased nuclear translocation of catalytically active PKC-zeta during mouse colonocyte hyperproliferation

Shahid Umar1, Joseph H. Sellin1,2, and Andrew P. Morris1,2

1 Department of Integrative Biology, Pharmacology, and Physiology, and 2 Department of Internal Medicine, Division of Gastroenterology, Hepatology, and Nutrition, University of Texas Health Science Center at Houston, Medical School, Houston, Texas 77030


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Protein kinase (PK) C-zeta is implicated in the control of colonic epithelial cell proliferation in vitro. However, less is known about its physiological role in vivo. Using the transmissible murine colonic hyperplasia (TMCH) model, we determined its expression, subcellular localization, and kinase activity during native crypt hyperproliferation. Enhanced mitosis was associated with increased cellular 72-kDa holoenzyme (PKC-zeta , 3.2-fold), 48-kDa catalytic subunit (PKM-zeta , 3- to 9-fold), and 24-kDa membrane-bound fragment (Mf-zeta , >10-fold) expression. Both PKC-zeta and PKM-zeta exhibited intrinsic kinase activity, and substrate phosphorylation increased 4.5-fold. No change in cellular PKC-iota /PKM-iota expression occurred. The subcellular distribution of immunoreactive PKC-zeta changed significantly: neck cells lost their basal subcellular pole filamentous staining, whereas proliferating cell nuclear antigen-positive cells exhibited elevated cytoplasmic, lateral membrane, and nuclear staining. Subcellular fractionation revealed increased PKC-zeta and PKM-zeta expression and activity within nuclei, which preferentially accumulated PKM-zeta . These results suggest separate cellular and nuclear roles, respectively, for PKC-zeta in quiescent and mitotically active colonocytes. PKM-zeta may specifically act as a modulator of proliferation during TMCH.

protein kinase C; cellular mitosis; mouse colon


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE INTESTINAL MUCOSA is a highly dynamic epithelium in which proliferatory cells within lower regions of the crypts migrate along a longitudinal axis to replace their senescent counterparts at the mucosal surface. Progression along this axis is associated with increased differentiation and phenotypic maturation. This process of mucosal homeostasis is normally tightly regulated to ensure that proliferative activity 1) balances the requirements for functionally mature cell types and 2) maintains mucosal integrity as senescent cells apoptose/exfoliate into the lumen. However, during mucosal hyperplasia and neoplasia a dramatic imbalance occurs, with proliferation predominating. Indeed, hyperproliferation and a concomitant shift in the number (hyperplasia) of immature colonocytes into compartments close to the mucosal surface are frequent cytokinetic abnormalities observed in the macroscopically normal mucosa of patients with colorectal neoplasia (33, 35, 42). Malignant transformation is a multistage process characterized both by activating mutations in protooncogenes (32) and by genetic alterations that inactivate tumor suppressor genes (45). In addition, accumulating evidence from both human (21, 23, 25) and animal (5, 15, 46) studies suggests that epigenetic changes in protein kinase (PK) C-dependent cellular signal transduction are integral to this process (reviewed in Ref. 48).

PKC is a multigene family consisting of at least 11 distinct lipid-regulated protein-serine/threonine kinases that play pivotal roles in signal transduction and growth control (8). Each isoform possesses unique structural properties as well as a distinct tissue and cell distribution, supporting the concept that individual isoforms perform specific roles in cellular signaling. The isoforms can be divided into three groups: 1) classical PKCs that are regulated by Ca2+ and diacylglycerol (DAG) (alpha , beta 1, beta 2, and gamma ), 2) novel PKCs that are Ca2+ independent but activated by DAG (delta , varepsilon , eta , theta , and µ), and 3) atypical PKCs (aPKCs) that are both Ca2+ and DAG independent (zeta , iota , and lambda ). A number of studies have shown a reduction in PKC activity in human colonic adenocarcinomas compared with normal adjacent mucosa (23, 25, 26). Furthermore, several laboratories using the procarcinogen 1,2-dimethylhydrazine (DMH) or its more proximate metabolite azoxymethane (AOM) to induce experimental colon cancer have demonstrated changes in PKC within both premalignant and malignant colonocytes. During the premalignant phase, translocation of PKC to the particulate fraction is observed, followed by subsequent decreases in the total biochemical activity (5, 15). It remains uncertain, however, whether initial activation or later downregulation is the most important PKC-signaling event promoting neoplastic change or whether these events reflect an unsuccessful attempt by these cells to negatively modulate proliferatory signals generated by activated oncogenic pathways. Because transmissible murine colonic hyperplasia (TMCH) has been identified as a condition that promotes DMH-induced carcinogenesis in the mouse (2, 4) and PKC-zeta signaling has been linked to cellular growth control, the escape of cells from apoptosis, and anchorage-independent growth (7, 8, 50, 52), we used this model to record the expression levels, location, and biochemical activation status of this kinase. Our results suggest a physiological role for PKC-zeta and, more importantly, its proteolytic fragment PKM-zeta in nuclear signaling events regulating hyperproliferation within the native colonic mucosa.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antibodies. Polyclonal rabbit anti-PKC-zeta anti-peptide antibodies were procured from Santa Cruz Biotechnology (SC-216/7282; Santa Cruz, CA) and Upstate Biotechnology (06-475; Lake Placid, NY) and were also received as a gift (C-14) from Dr. Todd Sacktor, State University of New York Downstate Medical Center (Brooklyn, NY) (see Table 1). Monoclonal anti-PKC-iota antibody was purchased from Transduction Laboratories (San Diego, CA), and proliferating cell nuclear antigen (PCNA) antibody was purchased from Signet Laboratories (Dedham, MA). FITC- and Texas red-conjugated antibodies for immunofluorescence studies were procured from Molecular Probes (Eugene, OR).

In vivo model for hyperplasia. TMCH was developed in Swiss-Webster mice (15-20 g; Harlan Sprague Dawley, Houston, TX) by oral inoculation with a 16-h culture of Citrobacter rodentium, formerly Citrobacter freundii biotype 4280 (3). Age- and sex-matched control mice received sterile culture medium only. Infected and normal mice were housed separately in microisolator cages in different rooms of the animal house facility. To determine the changes in the gross morphology of the Citrobacter-infected colonic mucosa, animals were killed by cervical dislocation after 2 wk of infection and their distal colons were removed, flushed with saline, embedded in OCT compound (Miles, IN), and cryopreserved in liquid nitrogen before sectioning and staining with hematoxylin and eosin.

Immunofluorescence. Distal colonic samples from normal and Citrobacter-infected animals were attached to a paddle and immersed in Ca2+-free standard Krebs buffered saline (in mM: 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 mM: 100 K-gluconate, 20 NaCl, 1.25 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, and 5 Na-pyruvate) and 0.1% BSA, resembling the intracellular medium. Suspended crypts were then deposited (1,200 rpm for 1 min) onto poly-L-lysine-coated microscope slides using a Shandon Cytospin cell preparation system. Immunolocalization studies were carried out by permeabilizing the crypts for 5 min in PBS containing 0.5% Triton X-100 (PBS-Triton). Nonspecific sites were blocked for 1 h with 3% BSA in PBS-Triton (blocking solution). Crypts were stained for the PKC-zeta isoform using polyclonal antibody diluted in blocking solution at 1:100 and 1:500, respectively. After incubation with appropriate antibodies either at room temperature for 1 h or at 4°C overnight, the slides were washed and incubated with goat anti-rabbit secondary antibody conjugated with either Texas red (PCNA) or FITC (PKC) and diluted in blocking solution at 1:500 for 1 h at room temperature or overnight at 4°C. Between washes, slides were incubated for 30 min in PBS containing 1% BSA. Control slides were incubated without the primary antibody or with isoform-specific primary antibody in the presence of appropriate antigenic peptide. Fluorescence was viewed using a Noran confocal laser scanning microscope (Noran Instruments, Middleton, WI) equipped with an argon laser and appropriate optics and filter modules for Texas red/FITC detection. Digital images 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 at 0.4-µm steps through the sample. Collected 8- or 16-bit color green-encoded images at 512 × 480 resolution were then stored on a mass storage device (removal rewriteable optical hard disk) and later volumetrically reconstructed using the Image-1/Metamorph 3-D module (Universal Imaging, West Chester, PA).

Preparation of nuclear extracts. Nuclear extracts were prepared from the distal colons of normal and Citrobacter-infected mice essentially as described by Zhang and colleagues (51) with some modifications. Briefly, tissues were cut and rinsed in saline A [in mM: 20 Tris · HCl, (pH 7.0), 137 NaCl, and 5 KCl] and homogenized in buffer A [in mM: 15 Tris · HCl (pH 7.0), 60 KCl, 15 NaCl, 2 EDTA, 0.5 EGTA, 1 dithiothreitol (DTT), 0.15 spermine, 0.5 spermidine, 0.4 PMSF, and 2 benzamidine], 0.25 M sucrose, and 1 µg/ml each of chymostatin, leupeptin, and pepstatin A. The homogenate was mixed with 2 vols of buffer B (buffer A with 2.3 M sucrose), layered on top of buffer C (buffer A with 1.8 M sucrose), and centrifuged at 25,000 rpm for 60 min at 4°C in an SW27 rotor. The nuclear pellets were resuspended in buffer D [in mM: 100 KCl, 10 Tris · HCl (pH 8.0), 2 MgCl2, 0.1 EDTA, 1 DTT, 0.4 PMSF, and 2 benzamidine and 1 µg/ml each of chymostatin, leupeptin, and pepstatin A]. The suspension was extracted with 0.1 vol of 4 M (NH4)2SO4 on a rotator for 30 min and then centrifuged at 30,000 rpm for 45 min in an SW40 rotor. The protein in the supernatant was precipitated with 0.3 g/ml (NH4)2SO4, pelleted, resuspended in buffer E [in mM: 20 HEPES (pH 7.8), 100 KCl, 0.2 EDTA, 0.5 DTT, 0.5 PMSF, and 2 benzamidine and 1 µg/ml each of chymostatin, leupeptin, and pepstatin A] and dialyzed against buffer E for 4-6 h. The dialysates were centrifuged for 5 min to remove the precipitates. Protein concentrations were determined, and extracts were used immediately for immunoprecipitation as described in PKC-zeta activity assay or frozen in liquid nitrogen and stored at -70°C.

Tissue preparation and Western blot analysis. Normal and Citrobacter-infected animals were killed by cervical dislocation 0, 1, 3, 6, 9, 12, and 15 days after infection, and their colons were removed and flushed with ice-cold PBS. Homogenates were prepared from whole distal colon as well as isolated crypts from a set of three animals for each infection in a buffer consisting of 50 mM Tris · HCl, pH 7.5, 0.25 M sucrose, 2 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 25 µg/ml each of leupeptin, aprotinin, and pepstatin, 1 µg/ml soybean trypsin inhibitor, 50 µM sodium fluoride, 50 µg/ml PMSF, and 10 mM beta -mercaptoethanol. The homogenate was centrifuged at 15,000 g for 15 min, and clear supernatant was saved as total tissue extracts. The soluble and particulate fractions from normal and Citrobacter-infected animals were prepared by homogenizing the whole distal colon or isolated crypts in the homogenizing buffer without Triton X-100. Cytosolic and membrane fractions were separated by sedimentation at 100,000 g in a centrifuge (TL-100, Beckman Instruments, Fullerton, CA) for 30 min. The resulting pellet was extracted in homogenizing buffer containing 1% Triton X-100, and the membrane subcellular fraction was recovered as the supernatant after centrifugation. Total tissue extracts or subcellular fractions (50-100 µg protein/lane) were subjected to 10% SDS-PAGE and electrotransferred onto the nitrocellulose membrane. Transfer efficiency was evaluated 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. Membranes were blocked with 5% nonfat dried milk in 20 mM Tris · HCl and 137 mM NaCl, pH 7.6 (TBS) for 1 h at room temperature and then at 4°C overnight. PKC isoforms were detected by incubating the nitrocellulose membranes for 2 h with anti-PKC isoform antibodies at 0.5-1.0 µg/ml in TBS containing 0.1% Tween 20 (TBS-Tween; Sigma Chemical). After being washed in TBS-Tween, membranes were incubated with horseradish peroxidase-conjugated goat-anti-rabbit IgG diluted 1:5,000 in 1% milk-TBS-Tween. After washes, immunoreactivity was detected using the enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL) according to the manufacturer's instructions.

PKC-zeta activity assay. PKC-zeta activity assay was carried out after immunoprecipitating either nucleus-free cellular or purified nuclear isolated crypt extracts from the distal colon of normal and infected mice. In brief, extracts were normalized for protein concentration and precleared for 1 h at 4°C with 30 µl of protein A-coated Sepharose beads. Immunoprecipitation was performed at 4°C by incubating the fractions for 2 h with polyclonal anti-PKC-zeta antibody and then for 1 h with 50 µl of protein A/G-Sepharose beads. Control experiments were performed by carrying out the immunoprecipitations in the presence of the immunizing peptides for PKC-zeta . After extensive washing, the immunoprecipitate was resuspended in 50 µl of a buffer containing 50 mM Tris · HCl (pH 7.5), 5 mM MgCl2, 1 mM PMSF, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM each of sodium orthovanadate, sodium pyrophosphate, and NaF. The kinase reaction for autophosphorylation was started by addition of 50 µM [gamma -32P]ATP at 37°C, and the phosphorylation was stopped by addition of 50 µl of reducing Laemmli sample buffer. Substrate phosphorylation was started by addition of 50 µM [gamma -32P]ATP at 37°C using PKC-varepsilon pseudosubstrate derivative (ERMRPRKRQGSVRRRV) as substrate for PKC-zeta in the presence or absence of inhibitor peptide. Stock solutions of lipids in ethanol and dilutions in kinase buffer were freshly prepared for each experiment from lyophilized aliquots. Phosphorylation was stopped by addition of reducing Laemmli buffer, and proteins were separated on 7.5% or 10% SDS-PAGE, transferred onto the nitrocellulose membrane, and analyzed by autoradiography as well as by Western blotting.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Establishment of model. TMCH is an infectious disease of mice caused by Citrobacter rodentium and characterized by significant epithelial cell proliferation within the mucosa of the descending colon with or without an inflammatory axis depending on the genetic background (3). In adult Swiss-Webster mice, crypt hyperplasia is not associated with significant changes in inflammatory status, and, at the time of the maximal hyperproliferatory response (2 wk after infection), this strain of bacteria no longer colonized the gut (1). Isolated distal colonic crypts from postinfection day 12 Citrobacter-inoculated mice showed increased abundance of PCNA (6-fold after normalization with beta -actin, n = 6 mice) compared with controls (Fig. 1A), and immunofluorescent PCNA staining within the normal crypt base was redistributed throughout the TMCH crypt axis (Fig. 1B). We have reported (43a) that apoptosis, a measure of programmed cell death, was similar in both normal and Citrobacter-infected crypts.


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Fig. 1.   Measurement of proliferating cell nuclear antigen (PCNA) abundance in cellular extracts of distal colonic crypts isolated from normal (N) and day 12 transmissible murine colonic hyperplasia (TMCH) (H) mice. A: Western blot analysis using anti-PCNA monoclonal antibody normalized to beta -actin (see MATERIALS AND METHODS). TMCH was associated with a 6-fold increase in cellular PCNA expression. B: in the same sample, the longitudinal axis of TMCH crypts revealed enhanced PCNA immunofluorescence staining (n = 7 individual experimental observations).

PKC-zeta abundance and activity changes occur during mucosal hyperproliferation. During TMCH, enhanced proliferative activity within colonic crypts is linked to selective activations in two [conventional (c) (PKC-beta 1) and novel (n) (PKC-varepsilon )] of seven phorbol ester-sensitive PKC isoforms detected in the colonic mucosa (43b). In this study, we sought to determine whether the expression levels and activation status of aPKC-zeta changed.

aPKC-zeta isoform expression in purified crypt extracts during TMCH. Colonic crypts express PKC-zeta (38, 44). The availability of isozyme-specific antisera for PKC-zeta detection prompted us to measure the expression levels of this aPKC.

Triton-solubilized extracts were prepared from isolated, purified crypts from animals 0, 1, 3, 6, 9, 12, and 15 days after Citrobacter infection and analyzed by Western blotting with a panel of isozyme-specific polyclonal anti-PKC-zeta antibodies. Specificity was determined by competitive blotting with corresponding immunizing peptides, and the protein concentrations were normalized by densitometry to beta -actin (to account for differences in gel loading). Basal levels of aPKC-zeta expression in isolated crypt extracts were similar to those of brain [ratio = 1.0 ± 0.1 (SD); n = 6 animals in duplicate] and were characterized by the presence of more than one immunoreactive species (Fig. 2).


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Fig. 2.   Mucosal hyperproliferation was associated with increased cellular atypical (a) protein kinase C (PKC)-zeta expression. Left, representative Western blot of isolated crypt cellular extracts collected from normal (N) and day 12 TMCH (H) mouse distal colon. Immunoreactivity was recorded at 72/76 (bands A, A*), 46/48 (bands B, B*), and 26/28 (bands C, C*) kDa. Right, blots made over the first 15 days after Citrobacter infection were stripped and probed for housekeeping protein beta -actin to create a bar graph of relative optical density (Rel. O.D.; each time point represents the mean ± SD of 2 or 3 experiments performed in duplicate). All immunoreactive bands increased their cellular expression during TMCH; some bands were barely detectable (A*, B*) or undetectable (C, C*) in normally proliferating crypts. Specificity was confirmed with a panel of anti-PKC-zeta specific peptide antibodies and their corresponding immunogenic peptides (Table 1).

The C-20 rabbit anti-rat PKC-zeta polyclonal antibody (Table 1) used for most of the Western blotting and immunofluorescence aspects of this study detected an immunospecific band at 72 kDa (band A, Fig. 2, left) which was accompanied by a very much fainter band at 76 kDa in hyperproliferating, but not normal, crypt extracts (band A*, Fig. 2, left). Additionally, immunoreactive doublets were recorded at 46/48 kDa and 26/28 kDa (bands B/B* and C/C*, Fig. 2, left). The relative expression of each band (~72, <48, and <29 kDa), increased significantly during TMCH (Fig. 2, right). Values quantified relative to beta -actin at postinfection day 12 at the time of the peak hyperproliferatory response were 72 (band A = 3.2-fold), 46 (band B = 3.3-fold), 48 (band B* = 9-fold), 26 (band C = >10-fold), and 28 (band C* > 10-fold) kDa (P < 0.01, n = 6 animals in duplicate). The time course of increased expression was the same for all bands. However, bands B*, C, and C* were only detected after postinfection day 6, and they continued to rise in parallel with bands A and B (Fig. 2, right). Band A* was too faint to detect on most gels and was not subjected to further analysis. Compared with crude brain homogenates run on the same gel (data not shown), overall levels of PKC-zeta holoenzyme were similar and hyperproliferating colonocyte PKM-zeta levels were 5.5-fold higher.

                              
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Table 1.   Summary of PKC-zeta band detection by the PKC-zeta peptide antibodies used for Western blotting

The authenticity of the immunodetected bands at 72, 48, and <29 kDa was confirmed by using a panel of different PKC-zeta antibodies together with the relevant competing peptide (Table 1). All three molecular mass species (72, 46/48, and 26/28 kDa) were recognized both by a goat anti-rat COOH-terminal polyclonal antibody that cross-reacts with murine PKC-zeta and the closely related lambda -isoform (C-20, Table 1) and by a more selective rabbit anti-rat COOH-terminal antibody (C-15, Table 1), with no reported cross-reactivity with cPKC-alpha or the murine aPKC-lambda /iota homologues (9, Table 1). However, band A* was not detected by a similar and closely related anti-rat PKC-zeta peptide antibody (C-14, Table 1) mapped to a slightly shorter region of the COOH-terminal domain (37). When extracts were probed with an NH2-terminal goat anti-human PKC-zeta selective antibody, non-cross-reactive with PKC-lambda (29), only the 72-kDa band was detected (N-17, Table 1). Immunocompetition with recombinant peptides against which C-20 and N-17 antibodies were made confirmed the immunospecificity of the 72-, 46/48-, and 26/28-kDa bands, respectively (Table 1). Further evidence supporting the notion that the 46/48-kDa immunoreactive species were closely related to the 72-kDa holoenzyme was found when using the rabbit C-14 anti-rat PKC-zeta COOH-terminal antibody, which has been shown to react with both PKC-zeta holoenzyme and its catalytically active 51-kDa proteolytic breakdown product, PKM-zeta (37). In this instance, only band A (72 kDa) and bands B and B* (46/48 kDa) were detected.

Because PKC-iota closely resembles PKC-zeta , we next used anti-PKC-iota antibody to check whether PKC-iota was contributing to PKM expression during TMCH. When nitrocellulose membranes probed with PKC-zeta C-20 antibody (Fig. 3A) were stripped and probed for PKC-iota with a monoclonal antibody specific for its catalytic domain, very modest (<1.1-fold, n = 3) increases in 72-kDa PKC-iota abundance were observed (Fig. 3B). However, no PKM-iota was detected in normal or hyperproliferating crypts; PKC-iota was not further investigated.


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Fig. 3.   Mucosal hyperproliferation did not affect PKC-iota expression. C-20 anti-PKC-zeta (A)- and anti-PKC-iota (B)-specific antibodies were used to probe normal (N) and day 12 (H) crypt cellular extracts by Western blotting (see MATERIALS AND METHODS). PKC-zeta immunoreactivity increased during TMCH, whereas PKC-iota expression exhibited only a very modest change. No detectable PKM-iota (bands B, B*) was recorded during TMCH.

The crypt epithelium therefore contained both PKC-zeta holoenzyme and PKM-zeta , together with a smaller immunoreactive species related to the COOH-terminal catalytic domain of PKC-zeta . Our studies with immunospecific cPKC and nPKC antibodies failed to resolve similar-molecular-weight bands (data not shown). The crypt-specific rise in aPKC-zeta cellular expression indicated that, along with cPKC-beta and nPKC-varepsilon isoforms (43b), PKC-zeta may participate in Citrobacter-induced colonocyte hyperproliferation.

Effect of mucosal hyperproliferation on PKC-zeta translocation. Because alterations in the subcellular compartmentalization of PKCs can be a surrogate and/or marker of activation, the distribution of PKC-zeta was assessed in the soluble (cytosolic) and particulate subcellular fractions of colonocytes at 12 days after Citrobacter infection. Analysis of the complex pattern of subcellular PKC-zeta partitioning by Western blotting of isolated crypt extracts from normal and day 12 Citrobacter-infected animals revealed (Fig. 4, left) that the membrane-to-cytoplasmic partitioning ratio (Rm:c) for PKC-zeta bands A and B/B* decreased during TMCH when PKC-zeta expression increased. Values relative to normally proliferating crypts were 2.4 ± 0.1-, 2.1 ± 0.1-, and 3.0 ± 0.2-fold lower, respectively, and were significantly different (Fig. 4, right; P < 0.05, n = 6 animals in duplicate). In contrast, band C and C* Rm:c increased >10- and 30-fold, respectively (Fig. 4, left). This complex pattern of decreased PKC-zeta holoenzyme and PKM-zeta membrane translocation together with almost complete band C membrane association was unexpected considering that movement of PKC from cytosol to intracellular membranes is assumed to predict activation. We therefore reasoned that increased cellular expression of PKC-zeta during TMCH was associated with the redistribution of this isoform to other locales within the cell.


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Fig. 4.   Mucosal hyperproliferation was associated with selective alterations in PKC-zeta immunoband membrane association. Left, representative Western blot of PKC-zeta immunoreactivity in membrane-to-cytosolic (m:c) fractions of normal (N) and day 12 TMCH (H) distal colonic crypts. Right, corresponding histogram of m:c partitioning ratio of PKC-zeta bands A (72 kDa), B/B* (46/48 kDa), and C/C* (26/28 kDa). Although the membrane association of bands A and B/B* decreased during TMCH, bands C/C* were almost entirely membrane associated (values were averaged from 3 independent experiments ± SD).

Nuclear PKC-varepsilon /-zeta partitioning during crypt hyperproliferation. Substantial evidence indicates a role for PKC in linking cell plasma membrane receptor signaling, particularly PKC-zeta , to events occurring at the genome level (10, 49). Because cellular mitosis is a nuclear event, activated PKCs involved in the regulation of colonic proliferation may redistribute into this organelle during TMCH. Purified nuclei and nucleus-free cellular fractions from normal and day 12 Citrobacter-inoculated mouse distal colons were probed for PKC-varepsilon and PKC-zeta expression using the anti-PKC-varepsilon and C-20 anti-PKC-zeta antibodies (Fig. 5).


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Fig. 5.   PKC-zeta is nuclear translocated in hyperproliferating crypts. Anti-PKC-varepsilon (A)- and C-20 anti-PKC-zeta (B)-specific antibodies were used to probe whole cell (lane 1), nuclear free supernatant (lane 2), and purified nuclear (lane 3) extracts by Western blotting (see MATERIALS AND METHODS). Significant PKC-varepsilon nuclear immunoreactivity was not detected. However, 72- and 46/48 kDa immunoreactive PKC-zeta bands were present in the nuclear extracts and levels were higher than in nucleus-free cellular extracts (similar amounts of protein were run on the gels). All bands were also competed out with 1:1 molar immunizing peptide (+P). C: when PKC-zeta immunoreactivity in purified nuclear extracts from normal (N) and day 12 TMCH (H) crypts were compared, the expression of all detected PKC-zeta immunobands increased. Hyperproliferating crypt nuclei accumulated more of the 46-kDa form of PKC-zeta (band B) than either 48-kDa (band B*) or 72-kDa (band A) forms. Band B was itself resolved into 2 separate bands on 7% gels (shown).

PKC-varepsilon immunoreactivity was barely detectable in Western blotted purified nuclear extracts in hyperproliferating mucosa (Fig. 5A). However, strong immunoreactivity was observed for PKC-zeta in hyperproliferating crypt nuclei probed with the C-20 antibody (Fig. 5B). Both PKC-zeta band A (72 kDa) and bands B*/B (46/48 kDa) were detected; band C (<29 kDa) was seen only faintly on overexposed gels. When similar amounts of protein were run on the gel, the levels of both PKC-zeta and PKM-zeta were higher in purified hyperproliferating crypt nuclei than in total cell extracts. This difference was most pronounced for PKM-zeta , in which nuclear levels were 2.2 ± 0.1-fold higher than total cellular levels (Fig. 5B). Specificity was confirmed by competitive blotting in the presence of immunizing peptide (Fig. 5, A and B). When nuclear levels of both PKC-zeta holoenzyme and PKM-zeta species were compared between normal and TMCH crypts, hyperproliferation was clearly associated with elevated nuclear accumulation of both bands A and B (Fig. 5C). On the same Western blots, an additional immunoreactive species running at 42 kDa was also recorded. These findings demonstrated that a substantial amount of both PKC-zeta and PKM-zeta was localized to the nucleus and that partitioning of these species into the nucleus accompanied the rise in cellular PKC-zeta and PKM-zeta expression recorded during TMCH (Fig. 2). However, these findings did not determine whether nuclear accumulated PKC-zeta or PKM-zeta was catalytically active. The same C-20 antibody was used to immunoprecipitate PKC-zeta in cellular extracts for the subsequent measurement of enzymatic activity.

Immunoprecipitable PKC-zeta enzyme activity. Activation of PKC-zeta is accompanied by intramolecular autophosphorylation (24, 43). To determine whether nuclear translocated PKC-zeta /PKM-zeta was catalytically active, autophosphorylation of PKC-zeta was measured in protein G-agarose-recovered immune complexes. Figure 6 is a representative blot showing both the immunoprecipitation protocol used and our control for C-20 anti-PKC-zeta antibody specificity.


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Fig. 6.   PKC-zeta immunoprecipitated from nucleus-free (A) and purified nuclear (B) fractions from normal (N) and day 12 TMCH (H) crypts exhibited subtle alterations in molecular mass. After immunoprecipitation with the C-20 anti-PKC-zeta antibody in the absence (-) or presence (+) of immunizing peptide, PKC-zeta immunoreactivity was detected by Western blotting with the same antibody. Although the apparent molecular mass of the 72-kDa band did not differ between fractions, the 48-kDa form of PKC-zeta preferentially accumulated in the nuclear-free cytoplasm and the 46-kDa form of PKC-zeta preferentially accumulated in the nucleus, which resolved as a doublet on 7% gels (shown). All PKC-zeta bands were competed away with immunizing peptide; the remaining signal () represents IgG heavy-chain fragments. This protocol was adapted for the measurement of intrinsic and extrinsic kinase activity.

After immunoprecipitation and transfer onto nitrocellulose membranes, PKC-zeta -immunoreactive bands A and B* were detected by Western blotting with the same antibody (Fig. 6). In nucleus-free cellular immunoprecipitates, bands recorded at 72 and 48 kDa were competed away with immunizing peptide (Fig. 6A, n = 6 animals in duplicate). In purified nuclear immunoprecipitates (Fig. 6B), bands were detected at 72 and 46/48 kDa that were likewise lost on competition with 1:1 molar immunizing peptide (n = 6 animals in duplicate). This immunoprecipitation protocol confirmed our earlier findings that both PKC-zeta holoenzyme and, more importantly, a PKM-zeta -like species were accumulated in hyperproliferating nuclei. This protocol was modified to measure the autophosphorylation status of the PKC-zeta holoenzyme (band A) and PKM-zeta (band B*/B) (Fig. 7).


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Fig. 7.   Modulation of PKC-zeta activity during TMCH. Left, PKC-zeta immunoprecipitated in nucleus-free cellular (A) and purified nuclear (B) fractions from day 12 TMCH crypts were used to estimate autophosphorylation levels (see MATERIALS AND METHODS). After nitrocellulose membrane autoradiography (i), PKC-zeta was Western blotted (ii). Intrinsic kinase activity corresponding to nonnuclear (Ai, 72 and 48 kDa) and nuclear (Bi, 72 and 46 kDa) forms of PKC-zeta were clearly detectable and were significantly reduced when the immunoprecipitation protocol was performed in presence (+) of immunizing peptide. Right, immunoprecipitated PKC-zeta from identical fractions was used to assay extrinsic kinase activity. Reconstituted kinase was incubated with modified PKC-varepsilon pseudosubstrate peptide (alanine to serine modification) in presence or absence of PKC-zeta inhibitor peptide and [gamma -32P]ATP (see MATERIALS AND METHODS). Substrate phosphorylating activity, calculated as picomoles per minute per milligram of protein, was found to increase 4.5- and 3.4-fold in the cellular and purified nuclear extracts, respectively (values are means ± SD from 3 individual experiments).

To detect intrinsic immunokinase activity, the immunoprecipitates were incubated with [gamma -32P]ATP and phosphatidylserine before SDS-PAGE and transfer to nitrocellulose membrane (see MATERIALS AND METHODS). Shown in Fig. 7 are autoradiographs of nucleus-free cellular and purified nuclear immunoprecipitates from TMCH crypts together with their corresponding Western blots (Fig. 7, left). Both sources exhibited active kinase at 72 kDa, corresponding to PKC-zeta holoenzyme. However, the intrinsic activity of bands B/B* was different in that signal was recorded at 48 kDa in nucleus-free cellular extracts and at 46/48 kDa in purified nuclear extracts. This difference was mirrored by PKM-zeta mobility differences on corresponding Western blots. Thus crypt hyperproliferation was accompanied by activation of PKC-zeta holoenzyme with an identical mobility in both subcellular compartments and compartment-specific PKM-zeta activation shown by subtle changes in gel mobility. The autophosphorylation levels of both bands were considerably reduced when immunoprecipitation was performed in the presence of the immunizing peptide (Fig. 7, left; n = 6 animals in duplicate). When the amount of autophosphorylation per zeta -isoform molecule was compared, 48-kDa cytoplasmic PKM-zeta was more autonomously active than lower-mass nuclear PKM-zeta . No difference was seen for the holoenzyme.

In addition to autophosphorylation, substrate phosphorylation of PKC-varepsilon pseudosubstrate peptide derivative by immunoprecipitated PKC-zeta was recorded (Fig. 7, right). TMCH was associated with a 4.5-fold increase in PKC-zeta activity in nucleus-free cellular extracts from isolated crypts (n = 3) and a corresponding 3.4-fold increase in PKC-zeta activity in nuclear extracts from the same source (n = 3, extracts made from day 12 TMCH animals). These studies confirmed the presence of catalytically active 72-kDa PKC-zeta and 46/48-kDa PKM-zeta in purified nuclear fractions isolated from hyperproliferating colonocytes.

Immunofluorescence staining of PKC-zeta . We next examined the immunolocalization of PKC-zeta in isolated crypts to understand whether increased cellular expression, decreased cytosolic membrane partitioning, and increased nuclear kinase activity during TMCH were paralleled by changes in PKC-zeta subcellular distribution (Fig. 8).


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Fig. 8.   Confocal volumetric reconstruction (100 × 0.4-µm z-axis planes) of cellular PKC-zeta staining in isolated crypts from normal (A) and day 12 TMCH (B). Crypts from both sources were simultaneously fixed, permeabilized, incubated with anti-PKC-zeta C-20 antibody (Table 1), and processed for immunofluorescence (see MATERIALS AND METHODS). Normal crypts (A) exhibited cellular staining predominantly within the neck region (arrow), whereas TMCH crypts (B) exhibited enhanced cellular staining throughout the crypt axis. Neither normal (C) nor hyperproliferating (D) crypts exhibited any signal when the primary antibody was omitted from the staining protocol. Digital contrast levels were not changed during image capture; crypts were isolated from 6 normal and 6 day 12 TMCH animals and were used for Figs. 9-11. Magnification, ×400.

Confocal microscopy with the C-20 PKC-zeta antibody (Table 1) and FITC-conjugated secondary antibody (see MATERIALS AND METHODS) revealed that PKC-zeta staining in volumetrically reconstructed normal mucosal crypts was highest in the apical and basolateral subcellular poles of colonocytes located within the crypt neck (Fig. 8A). In contrast, in hyperproliferating TMCH mucosa, subcellular PKC-zeta staining was evenly distributed throughout the length of the crypt (Fig. 8B). Concurrently processed crypts in which the primary antibody had been omitted failed to exhibit staining (Fig. 7, C and D). Because all crypts were processed and imaged identically, with no alteration in post-image-capture signal gain, changes in the overall levels of PKC-zeta subcellular staining in hyperproliferating crypts correlated very well with increased levels of cellular PKC-zeta expression reported by our Western blotting approaches (see Figs. 2, 3, and 6).

At higher magnification, crypts isolated from the normal mucosa also exhibited low levels of cytoplasmic PKC-zeta staining (Fig. 9). In this instance, PKC-zeta epithelium-specific cell labeling was confined to within 50 µm of the base of the normal crypt (Fig. 9A), corresponding to the region of PCNA immunostaining (Fig. 1), beyond which it diminished to background levels (Fig. 9A). In contrast, the elevated cytoplasmic PKC-zeta staining observed in hyperproliferating crypts did not vary greatly between these regions (Fig. 9B). Quantification revealed that the total (0.5 µm) z-axis plane immunofluorescent signal recorded 50 µm from the base of simultaneously processed normal and hyperproliferating crypts was 105 ± 15 and 218 ± 26 pixel intensities, respectively. At 100 µm from the base of the crypt, this signal decreased to 45 ± 5 pixel intensities in normal crypts but remained elevated at 197 ± 17 pixel intensities in hyperproliferating crypts (n = 20 individual crypts assayed in tandem from 6 mice). In addition, crypt surface-apposed bipolar cells of neuronal origin also exhibited PKC-zeta immunoreactivity (Fig. 9A). The latter cell population was recorded in all crypts at low frequency (2-4 cells/crypt) and was not addressed further. When quantified, control crypts for both conditions immunostained with FITC-conjugated secondary antibody and nonspecific IgG primary antibody exhibited low levels of background immunostaining (25 ± 18 pixel intensities, n = 60 crypts from 12 animals). Similar values were recorded when the primary antibody was omitted or when immunizing peptide was used to compete the primary antibody signal (data not shown).


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Fig. 9.   Higher-magnification (×800) confocal volumetric (100 × 0.4-µm z-axis plane) reconstructions of PKC-zeta immunoreactive staining in the basal cellular regions of normal (A) and day 12 TMCH (B) crypts. Simultaneous staining was performed; normal crypts exhibited weak cellular cytoplasmic immunoreactivity at their base, shown more clearly in the corresponding orthogonal z-y plane (Ai), but staining was considerably reduced 100 µm proximal to this region (Aii). In contrast, hyperproliferating crypts exhibited higher overall levels of cellular PKC-zeta staining at the crypt base (Bi) and in all regions proximal to this area (Bii). Crypt-apposed bipolar cells of neuronal origin were also present at low but equal density in both preparations (A). The staining pattern was eliminated by omitting the primary antibody. Asterisks indicate points along the longitudinal crypt axis where z-y images were taken.

Quantification of PKC-zeta immunofluorescent signal in the neck regions of normal and hyperproliferating crypts revealed another difference in PKC-zeta subcellular location (Fig. 10). Subcellular PKC-zeta immunofluorescence in colonocytes located within the neck region of normal crypts was evenly distributed between the subapical and subbasolateral cellular poles (Fig. 10A), whereas in hyperproliferating crypt neck regions PKC-zeta immunostaining was preferentially lost from the subbasolateral cellular region (Fig. 10B). Higher-spatial-resolution imaging (×800 magnification) within the neck region revealed that the basolateral subcellular pole staining in normal crypts was associated with as yet undefined filamentous subcellular structures (Fig. 11).


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Fig. 10.   Left, average PKC-zeta immunoreactive signal recorded from 3 confocal mid-z-axis (0.4 µm) x-y planes within the neck region of simultaneously processed normal (A) and day 12 TMCH (B) crypts. High levels of PKC-zeta staining were detected within the subcellular apical and basolateral poles of normal crypt colonocytes (cell position determined from crypt neck toward base, in A, 1right-arrow13). In contrast, hyperproliferating crypt neck regions exhibited decreased subcellular basal pole labeling (cell position in B, 1-9). Right, corresponding bar graphs of subcellular staining intensity collected with a 5.2-µm2 (26 × 26 bit) window placed over the apical (Ap), basolateral (Bas), and nuclear (Nuc) regions of individual cells within the neck region. Digitized fluorescence intensity values in this representative experiment are expressed as means ± SE for paired observations.



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Fig. 11.   Three representative single confocal planes of cellular PKC-zeta immunoreactivity in the neck region of the normal crypt at ×1,200 magnification. Planes were collected at -33 (A), 0 (B), and +35 (C) µm relative to the mid-z crypt axis. High levels of PKC-zeta immunofluorescent staining recorded at lower magnification in Figs. 7 and 9A were resolved as a filamentous subcellular staining pattern. In addition, infrequent bipolar cells of neuronal origin are stained.

Figure 11 shows three vertical 1-µm image planes taken at +5, +38, and +73 µm from the coverslip surface. Filamentous subcellular PKC-zeta immunostaining was most apparent in the vertical (z-axis) image plains close to the coverslip (+5 µm, Fig. 11A) and bath-apposed crypt surfaces (+73 µm, Fig. 11C), which bisect larger areas of subbasolateral plasma membrane cytoplasm. The mid-z-axis plane (+38 µm, Fig. 11B) revealed that filamentous immunostaining extended throughout the interior volume of the crypt. Very little nuclear staining was apparent.

When single x-y axis planes from the upper one-third or neck region of the TMCH crypts (12 days after Citrobacter infection) were analyzed at ×800 magnification, a different staining pattern was recorded (Fig. 12). Aggregation of PKC-zeta with subcellular basolateral microfilamentous structures was not as apparent. However, clear cytoplasmic, perinuclear, and nuclear PKC-zeta immunostaining was recorded (Fig. 12, A-C), as well as accumulation of PKC-zeta within the lateral but not basolateral plasma membrane (Fig. 12D). The nuclear accumulated PKC-zeta signal was clearly higher than that recorded in normal crypts, in which the nuclear volume was devoid of signal (Fig. 11B). Discrete hot spots within the hyperproliferating nuclei were detected (Fig. 12), suggesting aggregation of PKC-zeta into specific subnuclear structures. This pattern of PKC-zeta immunostaining was extended throughout the crypt axis and was similar to that recorded in the crypt base (n = 6 animals in triplicate).


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Fig. 12.   Representative single mid-z-axis confocal plane of PKC-zeta immunoreactivity recorded in the mid/neck region of a day 12 TMCH crypt. In images collected at ×800 magnification, diffuse cytoplasmic PKC-zeta immunoreactivity is evident, particularly within the subcellular apical pole (A, arrow). Areas within the same crypt at higher ×1,200 magnification (B-D) clearly showed prominent perinuclear and punctate nuclear immunoreactivity (B and D, arrow) together with accumulation of signal at or below the lateral plasma membrane (A and B, arrow-§). Note that hyperproliferating crypts failed to exhibit the same filamentous subcellular PKC-zeta staining as seen in the corresponding neck regions of normal crypts (Fig. 11).

Thus complex alterations in colonocyte subcellular PKC-zeta distribution that occurred as a consequence of crypt hyperproliferation were characterized as 1) loss from filamentous subbasolateral membrane cytosolic structures in crypt neck cells and 2) enhanced accumulation within the cytoplasm, at lateral plasma membrane and perinuclear sheaf, and within the nucleus. Thus the expression and subcellular distribution profile of PKC-zeta recorded in TMCH crypts corroborated the biochemical analysis of PKC-zeta expression and function outlined above (Figs. 2-6).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies have documented the distribution and biochemical activation status of PKC isoforms within the normally proliferating colonic epithelium. A gradient of PKC activity between crypt base and apex has been correlated with isoform-specific expression and subcellular location in an attempt to determine cellular function at different points along the crypt base-to-surface axis. Early studies reporting mass PKC activity found highest levels within the crypt base (14), whereas subcellular fractionation and immunological techniques revealed that most crypt-expressed PKCs are localized in the cellular cytoplasm in the lower proliferatory regions and only become membrane associated in mid to higher crypt regions (28). This leads to a translocation vs. activation paradox: PKC subcellular distribution in proliferatory crypt regions is opposite to that expected if membrane association predicts activation and an involvement in proliferatory growth control. However, this limited viewpoint may be an oversimplification of the true signaling status of the kinase because 1) it is based almost entirely on the PKC membrane-associating effects of phorbol esters (52), a model not directly relevant to atypical PKC activation; 2) it is becoming increasingly recognized that membrane association alone does not always predict substrate phosphorylation (52); and 3) PKCs exert their signaling effects at many locales within the cell through interactions with other proteins (29). Thus, in addition to participation in cell signaling cascades between the plasma membrane, internal membranes, and nucleus, direct PKC involvement in the nuclear responses of both mitogenic and differentiating factors was suggested recently (40). Our findings in the TMCH model directly address this question.

Cellular roles of aPKC-zeta in colonic epithelial proliferation. This isoform has received considerable recent interest. PKC-zeta has been implicated in the control of cellular mitogenic signaling and survival (16, 20, 34) and has been reported to play a pivotal role in tumor necrosis factor-alpha activation of nuclear factor-kappa B (17), an inducible transcriptional activator that participates in the control of cell proliferation and maintenance, as well as in inflammatory response and viral gene expression. The active component of fetal bovine serum-induced signaling in vitro, identified as lysophosphatidic acid, has also been shown to stimulate both Ras-dependent and -independent mitogen-activated protein (MAP) kinase cell signaling via phosphatidylinositol 3-kinase and aPKC-zeta activation (38). The role of this isoform as a downstream effector of p21ras, [RAS/RAF-MAP kinase kinase (MEK)-MAP kinase cascade] has been confirmed by the observations that PKC-zeta directly phosphorylates and activates MEK, resulting in the subsequent activation of MAP kinase (6, 12, 17, 39). Studies by Berra and colleagues (7) showed that the activation of PKC-zeta holoenzyme is critical for mitogenic signaling during maturation of Xenopus laevis oocytes (7).

Complex pattern of PKC-zeta expression and subcellular membrane partitioning accompanies TMCH. More than one immunoreactive PKC-zeta band was detected in normally proliferating and hyperproliferating isolated crypt extracts. Possible explanations for the appearance of multiple bands include the presence of closely related species or posttranslational modification of the parental holoenzyme. To address this concern, a panel of zeta -immunospecific antibodies were used. All three bands [holoenzyme, band A = 72 kDa; PKM-zeta , band B/B* = 46/48 kDa, and membrane-bound fragment (Mf)-zeta , band C/C* = 26/28 kDa] were recognized by two of three COOH-terminal antibodies, including one reported to be specific for PKC-zeta over the closely related PKC-lambda (9). The appearance of a catalytically active PKM-zeta fragment was noted previously by others after heterologous PKC-zeta overexpression in vitro (31). We hypothesize that band B* represents a closely related homologue or posttranslational modification to band B and that both B bands are proteolytic breakdown products of band A. In common with PKM-zeta , the immunoreactive <29-kDa PKC-zeta fragment routinely detected with COOH-terminal-specific antibodies (Fig. 2) was not observed when the NH2-terminal antibody was used for Western blotting (N-15, Table 1). Because our studies found that this lightest fragment was almost entirely membrane associated (Fig. 4) and was a fragment of the enzyme's catalytic domain, we designated this fragment as Mf-zeta .

Bands A and B exhibited markedly decreased membrane association during crypt hyperproliferation (Fig. 4, A/B) at a time when overall cellular levels of both species rose dramatically (Fig. 2), whereas band C was undetectable in normally proliferating crypts but, during TMCH, increased its expression in parallel with the other bands and was almost entirely membrane associated (Fig. 4C). Given that cellular levels of PKC-zeta increased in proliferating cells at a time when membrane-associated forms of this kinase were dramatically lowered, we investigated other possible cellular locales for the kinase. We found that in contrast to the majority of all other PKCs detected in crypts (43b), both PKC-zeta holoenzyme and PKM-zeta were nuclear translocated during TMCH (Figs. 5-7, 12). These observations indicated to us that either form of PKC-zeta , if catalytically active in the nucleus, could be hypothesized to play a direct role in regulating crypt colonocyte hyperproliferation.

Differences in PKM-zeta gel mobility correlated with nuclear accumulation of active PKM-zeta during TMCH. Phosphorylation has been found to be an important mechanism for regulating PKC activity. Two separate phosphorylation events appear to be required. The first event involves a critical threonine residue in the activation loop. This residue in PKC-zeta was recently shown to be phosphorylated in vivo by phosphoinositide-dependent protein kinase-1 (12, 27). After phosphorylation, autophosphorylation of the holoenzyme at critical serine and threonine residues within the catalytic domain occurs to fully activate the kinase (24, 43). To elucidate the activation status of PKC-zeta both within nucleus-free cytoplasm and within the nucleus of normal and TMCH colonocytes, immunoprecipitated enzyme from both subcellular pools was assayed for intrinsic kinase activity. TMCH increased the autophosphorylation status of both cytoplasmic and nuclear PKC-zeta /PKM-zeta , and these changes were reflected by increased varepsilon -peptide substrate phosphorylation in nuclear extracts. These in vivo findings were corroborated by previous reports that described small (8- to 10-fold; Ref. 36) to very large (200-fold; Ref. 11) increases in phospholipid-stimulated nuclear PKC activity after treatment of isolated nuclei with exogenous activators and by the fact that truncated forms of PKC encompassing the kinase domain are exclusively localized to the nucleus (18).

When related zeta -isoform Western blot and autophosphorylation data were compared, we found that protein expression and intrinsic kinase activity correlated for both nuclear and cytoplasmic holoenzyme and for cytoplasmic PKM-zeta . However, nuclear accumulated PKM-zeta paradoxically exhibited lower autonomous activity during TMCH, suggesting that some degree of inactivation had occurred. Because PKC phosphorylation leads to both activation and an increase in apparent molecular mass, the recorded decrease in PKM-zeta mobility on Western-blotted nuclear extracts may reflect this phenomenon. The function of zeta -isoform COOH-terminal domain autophosphorylation remains unknown. Our data suggests that posttranslational modification of nuclear PKM-zeta may be integral to modulation of the proliferatory signal during TMCH.

Changes in PKC-zeta subcellular distribution reflect biochemical changes in PKC-zeta activity recorded in TMCH crypts. Our immunofluorescence studies (Figs. 8-12) clearly demonstrated that crypt hyperproliferation was matched by enhanced immunoreactive PKC-zeta cell staining throughout the longitudinal axis of the crypt (Fig. 8) and by the accumulation of PKC-zeta signal within the cellular cytoplasm, at the nuclear envelope, and within the nucleus itself (Figs. 8, 9, and 12). Although there was incomplete overlap, PKC-zeta staining was observed in all crypt areas in which PCNA immunoreactivity (Fig. 1) occurred. Because the COOH-terminal antibody used for these studies detected all three PKC-zeta species (C-20, Table 1), we were unable to determine PKC-zeta /PKM-zeta /Mf-zeta specific subcellular location information. However, given that during TMCH only Mf-zeta increased its membrane association, whereas the other species exhibited decreased membrane translocation (Fig. 4), a significant fraction of the lateral membrane PKC-zeta immunoreactivity in TMCH crypts (Figs. 9 and 12) may be represented by this catalytically inert fragment. A corollary of this hypothesis was that PKC-zeta staining at this subcellular location represented a byproduct of enzyme activation/proteolysis and not active plasma membrane-associated kinase. Increases in nuclear envelope/nuclear staining recorded in TMCH crypts (Fig. 12) suggest that either PKC-zeta holoenzyme or PKM-zeta , but not Mf-zeta , was targeted to this location. In fact, both may achieve this through sequences within the COOH-terminal kinase domain (18).

Finally, the immunostaining pattern detected in normal and TMCH crypts differed in another respect. The neck regions of normal crypts exhibited clear cellular apical and basolateral pole immunostaining (Figs. 8 and 10) localized mainly to subcellular filamentous structures (Fig. 11). However, colonocyte hyperproliferation and the repopulation of this crypt region with immature cells resulted in the specific loss of the cellular basolateral pole staining pattern (Fig. 10B). It is interesting to speculate whether increased Mf-zeta expression (Fig. 2) reflects either the proteolysis of cytoskeletally aggregated or membrane-activated PKC-zeta and whether either of these cytoplasmic pools contributes to nuclear translocated PKC-zeta /PKM-zeta . It is possible that these different morphological findings reflect separate physiological roles for PKC-zeta in quiescent crypt neck and mitotically active crypt base colonocytes.

Model for subcellular PKC-zeta activation during TMCH. Figure 13 shows our working hypothesis of how during TMCH decreased levels of catalytically active, membrane-associated PKC-zeta /PKM-zeta (Figs. 4 and 6) can occur together with elevated cellular PKC-zeta activity (Fig. 7), PKC-zeta expression (Fig. 2), and enhanced cell division (Fig. 1). Differences in PKC-zeta holoenzyme, PKM-zeta , and Mf-zeta fragment expression and membrane association represent steady-state conditions present within hyperproliferating colonocytes. In the model described in Fig. 13, Mf-zeta describes a catalytically inert membrane proteolytic breakdown product of cytoplasmic holoenzyme PKC-zeta . Because activation of PKC and translocation to cell membranes is thought to be a prerequisite for proteolytic cleavage in vivo, PKM-zeta formed by the proteolysis of membrane-activated PKC-zeta (37) describes a constitutively active nuclear targeted kinase during TMCH. Subtle differences in gel mobility and autonomous activity between cytoplasmic and nuclear forms of autophosphorylated PKM-zeta identify postactivation cleavage of the PKC-zeta holoenzyme as an important regulatory component of nuclear PKM-zeta accumulation. However, at present we cannot also exclude the possibility that nuclear accumulated PKM-zeta may not have originated from the peripheral cytoplasm but rather was made within the endoplasmic reticulum and trafficked into the nucleus after initiation of translation from an internal ATG. Further studies are necessary to determine whether the activation of nuclear PKC-zeta /PKM-zeta preceded or followed nuclear translocation.


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Fig. 13.   Proposed model for subcellular PKC-zeta activation during TMCH. Mf, membrane-bound fragment.

Supporting our model, chronic activation of PKC-dependent mitogenic signaling in vivo has been shown to lead to increased cytoplasmic PKC expression (47), and, similarly, the in vivo effects of a number of mitogenic growth factors have been correlated with increased cytoplasmic PKC levels during periods of increased substrate phosphorylation (13, 22). Furthermore, chronic PKC-zeta activation during neuronal long-term potentiation has been correlated with loss of membrane-associated PKC-zeta and the cellular production of PKM-zeta (37). Thus we hypothesize that the translocation/activation paradox applied to PKC-zeta in hyperproliferating crypts can be explained by a switch from PKC-zeta holoenzyme signaling at subcellular membranes toward direct effects of PKM-zeta within the nucleus.

Physiological implications of PKC-zeta /PKM-zeta induction in hyperproliferating crypts. As outlined in the introductory paragraphs of this article, PKC-zeta has been implicated in colonic cancer progression. In both human and experimental colon cancers PKC-zeta expression has been shown to be decreased (52). Moreover, downregulation has been correlated with adenoma-to-carcinoma transition in AOM-treated mice. Subsequently, a variety of chemoprotective agents have been shown to preserve PKC-zeta expression in this and related carcinogenic models (48). In addition to the role of PKC-zeta in p21ras oncogenic signaling, PKC-zeta has also been reported to positively regulate the transcriptional activity of the tumor suppressor gene p53 in vitro and in vivo (44), mutations which prevent cells from initiating growth arrest or apoptosis. The loss of these latter regulatory mechanisms is also an established event in neoplastic transformation and provides cellular clues for another role of PKC-zeta in native colonocytes. In the TMCH model increased crypt cell number can be correlated with the fact that apoptosis fails to increase (43a) and thus match the sixfold enhancement in PCNA expression (Fig. 1). Therefore, in addition to regulating TMCH, alterations in PKC-zeta signaling may also correlate with the ability of mitotically active colonocytes to escape apoptotic cell death. Recent reports have implicated a prosurvival role of cellular PKC-zeta signaling, which must be inhibited for apoptosis to proceed (19). These phenomena, when coupled with other epigenetic signaling events, may be responsible for altering the phenotype of the hyperproliferative mucosa sufficiently to promote neoplastic transformation. This may explain why TMCH greatly reduces the time requirement for cancer formation in mice exposed to DMH/AOM and other DNA-hypermethylating colonic carcinogens (1, 4). Together, these studies imply that PKC-zeta may directly modulate gene expression during TMCH through signaling pathways that involve the activation of nuclear PKC-zeta /PKM-zeta . Efforts are underway in our laboratory to identify specific nuclear events and/or specific transcription factors associated with catalytically active PKC-zeta to understand the physiological mechanisms for Citrobacter-induced mucosal hyperproliferation at the genome level.


    ACKNOWLEDGEMENTS

This work was supported by funds from the Cystic Fibrosis Foundation and the American Institute for Cancer Research.


    FOOTNOTES

Address for reprint requests and other correspondence: Andrew P. Morris, Dept. of Internal Medicine, Division of Gastroenterology, Hepatology, and Nutrition, Univ. of Texas Health Science Center at Houston, Medical School, Houston, TX 77030 (E-mail: amorris{at}girch1.med.uth.tmc.edu).

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.

Received 9 November 1999; accepted in final form 1 February 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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