Activation of protein kinase C augments butyrate-induced differentiation and turnover in human colonic epithelial cells in vitro

Kurt L. Rickard1, Peter R. Gibson1, Graeme P. Young3 and Wayne A. Phillips2,4

1 University of Melbourne Department of Medicine, Royal Melbourne Hospital and
2 University of Melbourne Department of Surgery, Western Hospital, Melbourne, Victoria, Australia and
3 Department of Medicine, Flinders University of South Australia, Adelaide, Australia


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As the colonic epithelium is physiologically exposed to butyrate and to activators of protein kinase C, we examined the effect of the protein kinase C signalling pathway on butyrate-induced expression of markers of differentiation. Activators and inhibitors of protein kinase C were used in combination with butyrate and effects on the expression of markers of differentiation examined in colon cancer cell lines. When the protein kinase C activator phorbol myristate acetate (100 nM) was added for 24 h prior to the addition of 2 mM butyrate, there was a synergistic increase in alkaline phosphatase activity (154 ± 11% above that for butyrate alone, P = 0.003) in a concentration- and time-dependent manner. Butyrate-induced expression of carcinoembryonic antigen and interleukin-8, dome formation and cell turnover were also markedly augmented by pre-treatment with phorbol myristate acetate. A similar effect was observed with propionate or acetate (but not other differentiating agents), when phorbol myristate acetate and butyrate were added concurrently, or when other protein kinase C activators were used. Pharmacological inhibition of protein kinase C activity did not alter butyrate-induced alkaline phosphatase activity, but abrogated the augmentation induced by phorbol myristate acetate. We conclude that protein kinase C does not mediate the differentiating effects of butyrate on colon cancer cells, but its activation regulates butyrate-induced cellular differentiation.

Abbreviations: DiC8, 1,2-dioctanoyl-sn-glycerol; IL-8, interleukin-8; PdBt, phorbol 12,13-dibutyrate; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; OAG, 1-oleoyl-2-acetyl-rac-glycerol; SCFA, short chain fatty acids.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Short chain fatty acids (SCFA) play an important role in the welfare of the colonic epithelium, inducing a range of physiological effects (1). The principle SCFAs, acetate, propionate and butyrate, are produced in the colonic lumen as a consequence of anaerobic fermentation of undigested dietary carbohydrates. The SCFAs are taken up by colonocytes, where butyrate is preferentially oxidized as a fuel source (2). Experimental studies have demonstrated that butyrate improves intestinal epithelial barrier function in vitro (3) and promotes sodium absorption in vivo (4), while its absence from the colonic lumen leads to epithelial atrophy (5). Butyrate has also been shown to affect the morphology of cells, presumably by reorganization of cytoskeletal elements (6). Butyrate reduces proliferation of colorectal carcinoma cells in vitro via inhibition of DNA synthesis (68). Additionally, butyrate is a potent differentiating agent. It promotes the expression of a number of markers of cellular differentiation, including alkaline phosphatase and carcinoembryonic antigen, in colon cancer cells, inducing a phenotype typically associated with a normal mature cell (7,9). Recent studies have also demonstrated the ability of butyrate to promote apoptosis in a range of cell types at physiological concentrations (1013).

Despite the extensive body of knowledge regarding the effects of butyrate in biological systems, surprisingly little is known about the regulation of its effects or its mechanism of action, particularly at the signalling level. It has been demonstrated that butyrate-dependent effects on CHO cells can be inhibited by the protein kinase inhibitors H-7 and H-8 (14), whereas, in an erythroleukemia cell line (K562), butyrate has been reported to induce an increase in expression of protein kinase C (PKC) {alpha} and ßII isoforms (15). In addition, phorbol esters, which potently activate PKC, have been shown to produce cellular differentiative effects similar to those of butyrate. A range of butyrate-responsive cell lines differentiate in response to phorbol esters, including the malignant T lymphoblastic cell line MOLT-3 (16), K562 cells (15), the mouse myeloid cell line 32D (17) and the colorectal carcinoma cell lines SW 48 (18) and Caco-2 (19).

The differentiating effect of phorbol esters has been attributed to their role in activating PKC. It is well established that PKC plays an important role in the signal transduction mechanisms involved in regulation of cell growth, differentiation and apoptosis (20,21). Several members of this extended family of isoforms are potently activated by phorbol esters which may substitute for diacylglycerol, the physiological activator of PKC (21). In addition to activating PKC, treatment of cells with phorbol esters such as phorbol-12-myristate-13-acetate (PMA) (also referred to as 12-O-tetradecanoyl-13-acetate) and phorbol 12,13-dibutyrate (PdBt) also leads to the down-regulation of PKC (22,23). Phorbol esters have, therefore, become useful experimental tools for activating and depleting PKC in intact cells. Furthermore, colonic epithelial cells in vivo are exposed to activators of PKC, including secondary bile acids and diacylglycerols.

Given the range of processes influenced by both butyrate and the PKC signal transduction pathway and the normal concurrent exposure of colonic epithelial cells to butyrate and factors that activate PKC, it is of interest to know whether butyrate utilizes PKC in exerting its actions or, conversely, whether alterations in PKC status modulate the effects of butyrate on cell behaviour. The present study aimed, therefore, to investigate the interaction of butyrate and the PKC signal transduction pathway in the induction of biological effects of butyrate, principally cellular differentiation, in a range of colon carcinoma cell lines.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Unless otherwise noted, all chemicals were purchased from Sigma Chemical Co. (St Louis, MO). Ro 31-8220 (also known as bisindolymaleimide IX) was a generous gift from Dr G.Lawton (Roche Products, Welwyn Garden City, UK). Stocks of the phorbol esters, PMA, PdBt and 4{alpha}-phorbol, as well as the PKC inhibitors staurosporine and Ro 31-8220, were dissolved at 100 µM in DMSO and stored at –20°C until use. The final concentration of DMSO was 0.1% (v/v) or less and, at this concentration, DMSO alone did not have any detectable effects on indices measured. Sterile stocks of the SCFAs, acetate, propionate and butyrate, as well as 1,25-(OH)2 vitamin D3, all-trans retinoic acid and deoxycholate were prepared in phosphate-buffered saline, pH 7.4. The cell-permeable diacylglycerols, 1-oleoyl-2-acetyl-rac-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DiC8), were stored in chloroform at –20°C and immediately prior to use were evaporated to dryness under nitrogen gas and resuspended in phosphate-buffered saline (pH 7.4) with sonication and vortexing.

Cell culture
A number of colon cancer cell lines were utilized. The moderately differentiated LIM1215 colon cancer cell line (used between passages 20 and 35) and the highly differentiated HCT 8 cell line (passages 5–10) were kindly provided by Dr Robert Whitehead (Ludwig Institute, Melbourne, Australia). The well-differentiated Caco-2 human carcinoma cell line (passages 25–50) and the moderately differentiated HT-29 adenocarcinoma cell line (passages 30–40) were obtained from the American Type Culture Collection.

All cells were grown as monolayers and maintained in RPMI 1640 medium (Flow Laboratories, McLean, VA) supplemented with 10% fetal calf serum (Commonwealth Serum Laboratories, Parkville, Australia), 2 g/l NaHCO3, 4 mM glutamine, 20 mM HEPES, 50 mg/ml gentamicin and 50 U/ml penicillin. The cells were cultured at 37°C in an atmosphere of 5% CO2/95% air. Cells were maintained in 175 cm2 culture flasks (Nunc, Roskilde, Denmark) and were passaged by a 1 in 10 dilution of cells detached with 0.25% trypsin and 0.02% EDTA in phosphate-buffered saline (pH 7.4), for 3–5 min. For assay, cells were seeded at ~2x105 cells/ml in 6- or 24-well tissue culture plates (2 and 1 ml, respectively). The cells took 3–5 days to reach confluence with culture medium being changed every 1–2 days to avoid nutrient depletion. All cell culture ware was obtained from Becton Dickinson (Lane Cove, Australia).

Cell treatments
The effect of pre-treatment with a variety of PKC activators and inhibitors on butyrate-induced differentiation was examined. Unless otherwise indicated, LIM1215 cells were pre-treated with phorbol esters (100 nM) or diacylglycerols (10 µM) for 24 h prior to exposure to 2 mM butyrate for a further 48 h. The PKC inhibitors Ro 31-8220 (10 nM) and staurosporine (10 nM) were added for 1 h prior to addition of PMA or butyrate as appropriate.

Measurement of alkaline phosphatase activity and carcinoembryonic antigen levels
LIM1215 colon cancer cells were grown to confluence and incubated with the various compounds for the indicated times. Treated cells were washed twice in ice-cold phosphate-buffered saline, removed by scraping in mannitol buffer (50 mM D-mannitol in 2 mM Tris buffer, pH 7.4) and stored at –20°C until assayed. Immediately before assay, the cells were homogenized for 15 s using a Polytron homogenizer (Kinematica AG, Switzerland) and Triton X-100 added to the homogenates to 0.1% (v/v). Alkaline phosphatase activity in the cell homogenates was assayed spectrophotometrically according to the method of Young (24) using p-nitrophenol phosphate as substrate. Carcinoembryonic antigen levels were measured using a solid phase radioimmunoassay kit (Abbott CEA-RIA Diagnostic Kit no. 3A33-20; Abbott Laboratories, North Chicago, IL) according to the manufacturer's instructions. All values were adjusted for total cellular protein which was measured according to the method of Bradford (25) with bovine {gamma}-globulin as standard. All experiments were performed in quadruplicate. Alkaline phosphatase activity was expressed as mU/mg protein and carcinoembyronic antigen as ng/mg protein.

Assessment of domes
Confluent Caco-2 monolayers were grown on plastic 6-well plates and treated with 100 nM PMA for 24 h prior to the addition of 2 mM butyrate (for a further 48 h). The formation of domes is typical of transporting epithelial monolayers (26) and attributable to ion and water transport across functionally polarized epithelial cells (27). Dome formation was manually counted using phase-contrast light microscopy and expressed as no. domes/cm2.

Assay of lactate dehydrogenase activity
Lactate dehydrogenase leakage from cells was used as a measure of plasma membrane damage (28). LIM1215 monolayers were treated with appropriate stimuli for up to 72 h. After the incubation period, the culture medium was collected and centrifuged for 5 min at 1500 r.p.m. Supernatants (5 µl) were then treated with 250 µl lactate dehydrogenase reagent (Trace Scientific, Clayton, Australia), which contained 60 mM pyruvic acid, 0.23 mM NADH (isolated from yeast) and 55 mM phosphate buffer. Lactate dehydrogenase activity was determined spectrophotometrically by the decrease in absorbance at 340 nm using an Olympus AU5000 spectrophotometer (Olympus, Japan). All values were corrected for total cellular protein.

Cell number and [3H]thymidine uptake studies
The effect of various agents on the rate of DNA synthesis was determined by [3H]thymidine uptake. [3H]thymidine incorporation assays were carried out using 24-well tissue culture plates. LIM1215 cells were seeded at a density of 5x105 cells/well and allowed to attach for at least 24 h. Monolayers were then treated in a final volume of 0.5 ml of growth medium. A pulse of 15 µCi of methyl-[3H]thymidine (sp. act. 82 Ci/mM) was applied to the cells for the last 8 h of the incubation period (i.e. 40 h after the addition of the relevant agents). After this time, the cells were harvested onto glassfibre filters using a cell harvester (both obtained from Bartelt Instruments, Heidelberg West, Australia). After addition of 3 ml of non-aqueous Ready Value® scintillant (Beckman, Irvine, CA), [3H]thymidine incorporation into DNA was quantified in a Beta counter (LS 3801; Beckman, Irvine, CA). Uptake was expressed as disintegrations per minute (d.p.m.), relative to adherent cell number counted using a haemocytometer in parallel cell cultures. Cell viability was assessed by 0.4% trypan blue exclusion.

Measurement of apoptosis by DNA fragmentation
Confluent monolayers of LIM1215 cells were grown in 24-well tissue culture plates and treated at 37°C with the indicated agents for 72 h. Cells were then detached using a Tris buffer containing 0.25% trypsin and 0.02% EDTA, pH 7.4, and diluted with culture medium to obtain a final cell concentration of 105 cells/ml. Apoptosis was then assayed using a cell death detection ELISA according to the manufacturer's instructions (Boehringer Mannheim, Mannheim, Germany). This photometric enzyme immunoassay quantified cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) using mouse monoclonal antibodies directed against DNA and histones. The samples were analysed photometrically at 405 nm against a blank on a BioRad plate reader (BioRad, Richmond, CA) using Microplate Manager software (v.2.0.2). All experiments were performed in duplicate and results were expressed as a percentage of the control. The CV of the assay was <10%.

Measurement of interleukin-8 (IL-8) secretion
Secretion of IL-8 by LIM1215 cells was measured in cell supernatants using an in-house ELISA. Briefly, 100 µl of sample was added to microtitre plates pre-coated with anti-IL-8 polyclonal antibodies (R&D Systems, Minneapolis, MN) and incubated for 2 h at 25°C. Plates were washed and a monoclonal anti-IL-8 antibody (R&D Systems) was added to each well and incubated for a further 2 h at 25°C. Plates were washed again and an anti-mouse biotinylated immunoglobulin (DAKO) conjugate, diluted 1:20 000, added to each well. Plates were then incubated at 25°C for 90 min, washed and 100 µl of streptavidin–peroxidase, diluted 1:10 000 in dH2O, added to each well. Following a 30 min incubation at 25°C, plates were washed and 100 µl of tetramethylbenzidine added to each well. After a further 5 min the reaction was stopped by addition of 1 M phosphoric acid. The absorbances were read at 450 nm, against standards calibrated against World Health Organization reference material. Results were expressed as pg/mg. The CV of the assay was <10%.

Statistical analysis of data
Values are given as mean and standard error of at least three independent experiments. All statistical analyses were performed using Minitab® for Windows v.10.51 Xtra (Minitab, State College, PA). Where appropriate, differences between measurements were evaluated by two-tailed paired t-test using the raw data. Positive interaction was determined by analysis of variance using a general linear model. A P value <=0.05 was considered to be statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of butyrate and PMA on markers of differentiation
Exposure of LIM1215 cells to 2 mM butyrate for 24–72 h induced cellular differentiation as shown by an almost doubling of alkaline phosphatase activity (Figure 1Go). Exposure of the cells to PMA for 24 h also resulted in an almost doubling of alkaline phosphatase activity by 24–72 h after the PMA was removed from the cell cultures. Pre-treatment of the cells for 24 h with 100 nM PMA prior to the addition of butyrate resulted in an enhancement of the butyrate-induced alkaline phosphatase levels (Figure 1Go). This increase was observed at all time points after the addition of butyrate. At the 48 h time point PMA and butyrate were shown to positively interact (n = 12, P < 0.05).



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Fig. 1. Effect of pre-treatment with PMA on butyrate-induced changes in alkaline phosphatase activity. LIM1215 cells were pre-exposed to 100 nM PMA or carrier for 24 h. The culture medium was then removed and replaced with fresh medium alone or medium containing 2 mM butyrate. After the indicated times cells were washed and alkaline phosphatase activity measured in cell homogenates. Results are expressed as percent of control (without PMA or butyrate) at each respective time point. Shown are means ± SEM for five independent experiments. All data points for 24–96 h are significantly different (P < 0.05) from control (dashed line). *P < 0.05 compared with cells treated with PMA or butyrate alone (paired t-test).

 
Pre-exposure to PMA for 4–8 h was required before a significant enhancement of the effect of butyrate was detected. The magnitude of the enhancement increased with longer exposure to PMA up to 24 h (Figure 2AGo). The effect was also dependent upon the concentration of PMA, with a minimum of 1 nM PMA required for a significant effect increasing to a maximum at 100 nM (Figure 2BGo). Greater concentrations caused the cells to detach from the culture plates, indicating likely toxicity.



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Fig. 2. The effect of pre-treatment time and concentration on the enhancement of butyrate-induced alkaline phosphatase activity by PMA. (A) LIM1215 cells were pre-treated with 100 nM PMA for various times prior to exposure to 2 mM butyrate for a further 48 h. (B) Cells were pre-treated with various concentrations of PMA for 24 h prior to exposure to 2 mM butyrate for a further 48 h. Alkaline phosphatase activity was expressed as percent of control (no PMA or butyrate). Shown are means ± SEM for (A) five and (B) four independent experiments. *P < 0.05 compared with cells treated with butyrate alone (paired t-test).

 
The interaction between butyrate and PMA was also dependent upon the concentration of butyrate with a maximal effect seen at 2–3 mM butyrate (Figure 3Go). Exposure to higher concentrations of butyrate (4 mM), whether pre-treated with PMA or not, resulted in detached cells indicative of toxicity.



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Fig. 3. Enhancement of butyrate-induced alkaline phosphatase activity by PMA; effect of butyrate concentration. LIM1215 cells were pre-treated with 100 nM PMA (or carrier) for 24 h before exposure to various concentrations of butyrate for a further 48 h. Results are expressed as percent of control (no PMA or butyrate). Shown are mean ± SEM for five independent experiments. *P < 0.05 compared with butyrate-treated cells not pre-exposed to PMA (paired t-test).

 
At the concentrations used, the synergism between PMA and butyrate required the removal of PMA before the addition of butyrate. Co-stimulation of LIM1215 cells with 2 mM butyrate and 100 nM PMA did not significantly enhance alkaline phosphatase levels above that with butyrate alone (Figure 4AGo). Indeed, microscopic observation of the cells suggested that the combination of butyrate and PMA together for 48 h was inducing cell death. This was confirmed by the demonstration of a significant elevation in lactate dehydrogenase in the supernatant of these cells compared with the supernatant from untreated control cells or cells pre-treated with PMA alone (Figure 4BGo). However, co-treating cells with butyrate and a lower concentration of PMA (10 nM) did induce a significant enhancement of alkaline phosphatase activity, without a corresponding rise in supernatant lactate dehydrogenase activity (Figure 4Go).



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Fig. 4. Effect of PMA on butyrate-induced alkaline phosphatase activity and LDH release; pre-treatment compared with co-treatment. LIM1215 cells were either pre-treated with 100 nM PMA for 24 h and removed prior to addition of 2 mM butyrate or PMA (100 or 10 nM) was added together with butyrate (2 mM). After 48 h exposure to butyrate and/or PMA the culture medium were collected and the cells washed and scraped into mannitol buffer. Alkaline phosphatase activity in the cells (A) and lactate dehydrogenase levels in the culture medium (B) were assessed as described in Materials and methods. Results were expressed as percent of control (no PMA or butyrate). Shown are means ± SEM for four independent experiments. *P < 0.05 compared with control cells treated with butyrate alone (paired t-test).

 
The interactive effects of butyrate and PMA were also detected in other colon cancer lines. PMA pre-treatment potentiated butyrate-induced alkaline phosphatase activity in Caco-2, HT-29 and HCT-8 cells. The degree of enhancement was similar across all four cell lines even though butyrate alone had variable effects on alkaline phosphatase activity (Figure 5Go). Furthermore, PMA also potentiated the effects of butyrate on other markers of differentiation. Pre-treatment of LIM1215 cells with PMA resulted in a synergistic enhancement in carcinoembryonic antigen levels (Table IGo). The effect was also observed on dome formation in Caco-2 cells (Table IGo).



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Fig. 5. Effect of PMA pre-treatment on butyrate-induced alkaline phosphatase activity in colon cancer cell lines. LIM1215, Caco-2, HT-29 and HCT-8 cells were exposed to 100 nM PMA for 24 h prior to exposure to 2 mM butyrate (or carrier) for a further 48 h. Results are expressed as percent of control (no PMA or butyrate). Shown are means ± SEM for five independent experiments. *P < 0.05 compared with cells not treated with PMA or butyrate (paired t-test).

 

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Table I. The effect of PMA pre-treatment on butyrate-induced changes in markers of cellular differentiation
 
Effect of SCFAs, other differentiating agents and PMA on markers of differentiation
Interestingly, the potentiating effects of PMA appear to be specific for differentiation induced by SCFAs. Although acetate and propionate at a concentration of 2 mM had little effect on alkaline phosphatase activity of LIM1215 cells, whether pre-treated with PMA or not, at higher concentrations (8 mM propionate or 16 mM acetate) alkaline phosphatase activities were significantly increased. In PMA-treated cells, alkaline phosphatase activities were significantly greater than those seen with either SCFA alone (proprionate alone, 179 ± 33% of control cells, propionate on PMA-pre-treated cells, 367 ± 108%, P < 0.05; acetate alone, 170 ± 22%, acetate on PMA-pre-treated cells, 312 ± 96%, P < 0.05). However, pre-treatment of LIM1215 cells with PMA did not potentiate the induction of alkaline phosphatase activity by 2% DMSO, 10 nM 1,25-(OH)2 vitamin D3 or 10 mM all-trans retinoic acid (Table IIGo).


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Table II. The effect of PMA pre-treatment on changes in alkaline phosphatase activity induced by various differentiating agents
 
Effect of butyrate and PMA on the rate of DNA synthesis, apoptosis and on IL-8 secretion
As shown in Figure 6Go, butyrate (2 mM) induced a 3-fold increase in DNA fragmentation in LIM1215 cells, compared with control cells (P = 0.015). Treatment of cells with PMA alone did not influence apoptosis compared with controls, but exposure of cells to the phorbol ester prior to butyrate caused a 6-fold enhancement of butyrate-induced cell death compared with controls (P = 0.012) and doubling that of butyrate alone (P = 0.03). Although PMA alone caused a significant increase in [3H]thymidine incorporation compared with controls (P = 0.003), pre-exposure of cells to the phorbol ester actually augmented the anti-proliferative effect of butyrate by >20% (Figure 6Go). Secretion of IL-8 from LIM1215 cells was stimulated by both butyrate and PMA. Pre-treatment of the cells for 24 h with PMA prior to the addition of butyrate induced a 3-fold elevation of IL-8 secretion above that of butyrate alone (IL-8 secretion by LIM1215 cells: untreated, 100%; 2 mM butyrate alone, 476 ± 53%; 100 nM PMA alone, 431 ± 62%; PMA + butyrate, 1293 ± 163%; mean ± SEM, n = 4).



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Fig. 6. Effect of pre-treatment with PMA on butyrate-induced changes in cell turnover. LIM1215 cells were exposed to 100 nM PMA for 24 h prior to exposure to 2 mM butyrate for a further 48 h. DNA fragmentation and proliferation are expressed as percent of control (no PMA or SCFAs). Shown are the means ± SEM for three (apoptosis) and six (proliferation) independent experiments. *P < 0.05 compared with control cells and **P < 0.05 compared with all other treatments (paired t-test).

 
Studies of the mechanism of the interaction between butyrate and PMA
Role of the activation of PKC.
Treatment of cells with PMA results in an initial activation of PKC followed by its down-regulation. Thus, the synergistic effect of PMA could potentially occur as the result of the initial activation of PKC or of its subsequent depletion. To examine the role of PKC activation, two approaches were taken. First, the effects of pre-incubation with other activators of PKC prior to exposure to butyrate were examined. As shown in Figure 7Go, pre-treatment of LIM1215 cells with five known activators of PKC (PMA, PdBt, DiC8, OAG and deoxycholate) all induced a 2- to 4-fold elevation of butyrate-induced alkaline phosphatase activity above that of butyrate alone. In contrast, 4{alpha}-phorbol, which does not result in direct activation of PKC, had no effect on alkaline phosphatase activity alone or in combination with butyrate (Figure 7Go).



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Fig. 7. Effect of pre-treatment with various PKC activators on butyrate-induced alkaline phosphatase activity. LIM1215 cells were pre-treated with 100 nM PMA, 100 nM PdBt, 100 nM 4{alpha}-phorbol (4{alpha}), 10 µM DiC8, 10 µM OAG or 100 nM deoxycholate (DOC) for 24 h prior to exposure to 2 mM butyrate or carrier for 48 h. Results are expressed as percent of control (no PMA or butyrate). Shown are means ± SEM for five independent experiments. *P < 0.05 compared with butyrate-treated cells not pre-exposed to PKC agonists (paired t-test).

 
The role of depletion of PKC activity.
In order to simulate PKC depletion, LIM1215 cells were treated with the PKC inhibitor Ro 31-8820 (10 nM) for 1 h prior to the addition of 100 nM PMA and/or 2 mM butyrate. As shown in Figure 8Go, pre-treatment with Ro 31-8820 eliminated the effect of PMA but did not alter alkaline phosphatase activity induced by butyrate alone. Similar results were also observed using a second PKC inhibitor, staurosporine (data not shown).



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Fig. 8. Effect of pre-treatment with Ro 31-8220 on PMA- and butyrate-induced alkaline phosphatase activity. LIM1215 cells were treated with and without 10 nM Ro 31-8220 (Ro) for 1 h prior to treatment with 100 nM PMA for 24 h. The medium was then removed and replaced with fresh medium containing 2 mM butyrate for a further 48 h. Results are expressed as percent of control (no Ro 31-8220, PMA or butyrate). Shown are means ± SEM for four independent experiments. *P < 0.05 compared with butyrate-treated cells not pre-exposed to PMA or Ro 31-8220 (paired t-test).

 
Role of histone deacetylase.
Since butyrate can inhibit histone deacetylase causing marked changes in expression of a range of genes, cells pre-treated with and without PMA were exposed to trichostatin A, a potent and specific histone deacetylase inhibitor. Treatment of LIM1215 cells with trichostatin A caused a significant increase in alkaline phosphatase activity compared with controls (35.8 ± 4.4% increase; n = 4, P = 0.021). In cells pre-treated with PMA, trichostatin A induced a 208 ± 30% increase in alkaline phosphatase activity which was significantly greater than that in cells not treated with trichostatin A (P = 0.006). These effects were comparable with those of butyrate in parallel experiments (data not shown).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Like butyrate, PMA was found to elevate levels of the differentiation markers alkaline phosphatase and carcinoembryonic antigen in LIM1215 cells and dome formation in Caco-2 cells (Table IGo). More interestingly, pre-treatment with PMA was found to dramatically potentiate the differentiating effects induced by subsequent exposure to butyrate. This enhancement was time and concentration dependent for both butyrate and PMA. In addition to butyrate, PMA also enhanced the alkaline phosphatase activity induced by the two other physiologically relevant SCFAs, acetate and propionate, although to a lesser extent than seen with butyrate. This difference in the relative potencies of the SCFAs has been observed with regard to other SCFA-mediated effects in colon cancer cell line monolayers, such as increasing transepithelial resistance (3) or promoting migration following wounding (29). It may be related in part to their absorption by the cell, as the lipid solubility of the SCFAs decreases with chain length (30). The effect of PMA pre-treatment appears to be a specific effect of PMA on the differentiating pathway induced by SCFAs since the augmentation of alkaline phosphatase activity was not seen for the other `differentiating agents', DMSO, vitamin D3 or retinoic acid. The synergistic effect of PMA and butyrate was also observed during co-exposure with the two agents, although the concentration of PMA had to be reduced to avoid induction of cell death, consistent with previous observations in the VACO 5 and COLO 201 colon cancer cell lines (31,32).

The interaction effect of PMA and butyrate was also observed for other biological effects of butyrate. Thus, as previously reported in other cell lines (31,32), butyrate-induced apoptosis was markedly enhanced by treating cells with PMA prior to the addition of butyrate. Furthermore, the ability of butyrate to increase the secretion of IL-8 was markedly enhanced by treating cells with PMA prior to the addition of butyrate and the inhibiting effect of butyrate on DNA synthesis was exaggerated in PMA pre-treated cells, despite the action of PMA alone in stimulating DNA synthesis. Taken together, these results indicate that a wide range of butyrate-dependent cellular effects can be significantly enhanced by the activation of PKC prior to the addition of the SCFA.

Phorbol esters are potent and well-characterised activators of PKC and it is generally believed that this enzyme mediates the biological actions of PMA (21). Consistent with this, other activators of PKC, including the diacylglycerols DiC8 and OAG and the phorbol ester PdBt, were also found to potentiate the butyrate-induced increase in alkaline phosphatase activity. Similarly, the bile salt deoxycholate, which has been demonstrated to activate PKC (33,34), also synergized with butyrate to enhance alkaline phosphatase activity. In contrast, 4{alpha}-phorbol, a phorbol ester which does not activate PKC, failed to enhance the effects of butyrate. In addition, the potentiating effects of PMA could be suppressed by the PKC inhibitors Ro 31-8220 and staurosporine. Together these results provide strong support for the contention that PKC is involved in modulating the action of butyrate in colon cancer cells.

Since activation of PKC leads to its down-regulation, prolonged exposure to potent activators of PKC (such as PMA) results in depletion of PKC from the cells (22,23). The requirement for pre-treatment of cells for several hours before obtaining significant potentiation of butyrate-induced differentiation is consistent with the time course for the down-regulation of several PKC isotypes (23). However, unlike PMA pre-treatment, inhibition of PKC with the inhibitor Ro 31-8220 or staurosporine had no effect on butyrate-induced differentiation. This would imply that it is not the loss of PKC activity resulting from down-regulation which is responsible for the potentiating effects of PMA in this system. Rather it would appear that it is the initial activation of PKC that is important. Certainly, inhibition of PKC activity by Ro 31-8220 or staurosporine suppressed the potentiating effects of PMA pre-treatment.

The precise mechanism by which activation of PKC influences the action of butyrate is not clear. The fact that PKC inhibitors did not suppress the differentiation induced by butyrate alone would suggest that butyrate does not directly act via the PKC pathway. Although PMA results in rapid activation of PKC, a prolonged pre-treatment period was required to achieve optimal augmentation of the effects of butyrate. This would suggest that events such as new protein synthesis or modulation of gene transcription are involved in the interaction. Butyrate influences the expression of many genes and one mechanism proposed for such an action is inhibition of histone deacetylase (35,36). This leads to hyperacetylation of DNA, permitting increased exposure of DNA to transcription factors (37). Inhibition of histone deacetylase with a specific inhibitor, trichostatin A (38), mimicked the effects of butyrate in inducing the increased activity of alkaline phosphatase in LIM1215 cells and this was greatly enhanced by pre-treatment of cells with PMA. Activation of PKC has been implicated in the regulation of several transcription factors, including, AP-1 and NF{kappa}B (39). Potentially, therefore, the interactive effect of PMA and butyrate may result from butyrate-induced changes in histone acetylation leading to increased exposure of DNA to transcription factors stimulated by PKC activation. The role of the histone acetylation pathway in mediating the effects of PMA is also suggested by the observation that other `differentiating' agents, such as DMSO, vitamin D3 and retinoic acid, which do not induce changes in histone acetylation (40,41), do not synergize with PMA.

In this study, we have not attempted to address the in vivo relevance of our findings. However, an interaction of butyrate and other SCFAs with activators of PKC within the colonic epithelium is likely. SCFAs and PKC activators, such as diacylglycerols and deoxycholate, are both found normally within the lumen of the colon (34,42), where they have access to colonic epithelial cells. Since concurrent exposure of cells in vitro to PMA and butyrate also leads to synergistic effects in a key cellular process such as differentiation, the interaction of butyrate and PKC activators in vivo is, therefore, of potential importance for the biology of the colon. The interaction, for example, exaggerates antitumour effects such as the promotion of differentiation or induction of apoptosis of mutated cells and butyrate antagonizes the pro-tumourigenic effect of PKC activation on cell proliferation. These issues highlight the possibility that this interaction may be an appropriate target for dietary and/or pharmacological intervention in the treatment or prevention of diseases of the large bowel.

In conclusion, this study indicates that PKC plays an important role in regulating many of the biological effects of butyrate, including differentiation and cell turnover. It is the activation, not depletion of PKC, that appears to prime the cells to dramatically enhance their responsiveness to butyrate. However, since inhibition of the kinase does not change the effects of butyrate alone on alkaline phosphatase activity, it would seem unlikely that butyrate is directly acting via a PKC-mediated pathway.


    Acknowledgments
 
K.L.R. was a recipient of a Postgraduate Scholarship from the Gastroenterological Society of Australia.


    Notes
 
4 To whom correspondence should be addressed at: Department of Surgery, Western Hospital, Footscray, Victoria 3011, Australia Email: phillips{at}medicine.unimelb.edu.au Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 3, 1998; revised November 3, 1998; accepted February 2, 1999.