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Originally published In Press as doi:10.1074/jbc.M201747200 on May 1, 2002

J. Biol. Chem., Vol. 277, Issue 29, 26310-26320, July 19, 2002
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Expression of Endomembrane Calcium Pumps in Colon and Gastric Cancer Cells

INDUCTION OF SERCA3 EXPRESSION DURING DIFFERENTIATION*

Pascal GélébartDagger §, Tünde Kovács||, Jean-Philippe Brouland**, Roosje van GorpDagger , Johannes GrossmannDagger Dagger , Nathalie Rivard§§, Yves Panis¶¶, Virginie MartinDagger , Raymonde BredouxDagger , Jocelyne EnoufDagger , and Béla PappDagger ||||

From the Dagger  Unité 348 INSERM, IFR-6, Hôpital Lariboisière, 75010 Paris, France,  National Institute of Haematology and Immunology, Daròczi ùt 24, 1113 Budapest, Hungary, ** Service d'Anatomie Pathologique, Hôpital Lariboisière, 75010 Paris, France, Dagger Dagger   Klinik und Poliklinik für Innere Medizin I, Klinikum der Universität Regensburg, 93042 Regensburg, Germany, §§ Faculté de Médecine, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Québec J1H 5N4, Canada, and ¶¶ Service de Chirurgie Générale et Digestive, Hôpital Lariboisière, 75010 Paris, France

Received for publication, February 20, 2002, and in revised form, April 30, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Calcium mobilization from the endoplasmic reticulum (ER) into the cytosol is a key component of several signaling networks controlling tumor cell growth, differentiation, or apoptosis. Sarco/endoplasmic reticulum calcium transport ATPases (SERCA-type calcium pumps), enzymes that accumulate calcium in the ER, play an important role in these phenomena. We report that SERCA3 expression is significantly reduced or lost in colon carcinomas when compared with normal colonic epithelial cells, which express this enzyme at a high level. To study the involvement of SERCA enzymes in differentiation, in this work differentiation of colon and gastric cancer cell lines was initiated, and the change in the expression of SERCA isoenzymes as well as intracellular calcium levels were investigated. Treatment of the tumor cells with butyrate or other established differentiation inducing agents resulted in a marked and specific induction of the expression of SERCA3, whereas the expression of the ubiquitous SERCA2 enzymes did not change significantly or was reduced. A similar marked increase in SERCA3 expression was found during spontaneous differentiation of post-confluent Caco-2 cells, and this closely correlated with the induction of other known markers of differentiation. Analysis of the expression of the SERCA3 alternative splice isoforms revealed induction of all three known iso-SERCA3 variants (3a, 3b, and 3c). Butyrate treatment of the KATO-III gastric cancer cells led to higher resting cytosolic calcium concentrations and, in accordance with the lower calcium affinity of SERCA3, to diminished ER calcium content. These data taken together indicate a defect in SERCA3 expression in colon cancers as compared with normal colonic epithelium, show that the calcium homeostasis of the endoplasmic reticulum may be remodeled during cellular differentiation, and indicate that SERCA3 constitutes an interesting new differentiation marker that may prove useful for the analysis of the phenotype of gastrointestinal adenocarcinomas.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cellular calcium concentration gradients and calcium ion fluxes are important components of several signaling networks controlling cell growth, differentiation, or apoptosis (1, 2). In a resting cell, the cytosolic free calcium concentration is ~50-100 nM, whereas the endoplasmic reticulum (ER)1 or the extracellular medium contains calcium in the high micromolar to low millimolar range. Binding of several growth factors, hormones, chemokines, or bioactive peptides to their cell surface receptors leads to the formation of the second messenger inositol 1,4,5-trisphosphate (IP3), which induces calcium release from the endoplasmic reticulum into the cytosol through IP3 receptor calcium channels. The ensuing decrease of the calcium content of the ER lumen induces the opening of calcium channels in the plasma membrane, allowing calcium influx into the cytosol from the extracellular space. Calcium release from the ER and ensuing calcium influx lead to the augmentation of the cytosolic free calcium concentration. As many key components of intracellular signaling networks, such as various calmodulin-activated kinases (3), protein kinase C (4), calcineurin (5), calpains (6), as well as the PYK-2 tyrosine kinase (7), the Ras guanine nucleotide exchange factor Ras-GRF (8), the apoptosis-associated kinase DAP-2 (9), or the apoptosis-linked calcium-binding protein ALG-2 (10), are directly activated by increased cytosolic calcium concentrations, cellular calcium fluxes constitute an important component of several signal transduction networks of the cell.

In addition to its role played in signaling in the cytosolic compartment, calcium stored within the ER lumen is required for the post-translational modification and processing of newly synthesized proteins transiting across the organelle (11). It is becoming increasingly clear that calcium stored in the ER is involved in homeostatic, synthetic, as well as signaling functions also within the lumen of the organelle (12-14).

Refilling of calcium into the ER from the cytosol by active ATP-driven ion transport is ensured by sarco/endoplasmic reticulum calcium transport ATPases, also called SERCA enzymes (12). These enzymes, by pumping calcium into the ER against a steep concentration gradient, decrease cytosolic calcium levels after an episode of activation and make calcium available in the ER lumen for intra-ER calcium-dependent functions, as well as for being released into the cytosol during a next signaling event. Three SERCA genes are known, which by alternative splicing can give rise to several protein isoforms. SERCA1a and -1b are expressed in adult and neonatal skeletal muscle, respectively (15). SERCA2a is found in cardiac and smooth muscle, whereas SERCA2b has been found in all non-muscle cell types studied so far (16, 17). Expression of SERCA3 has been detected in a selected group of cell types, including cells of hematopoietic origin, where this enzyme is constitutively expressed (18-21), and the existence of three SERCA3 alternative splice isoforms has been reported (22-24). Although SERCA3 mRNA has been detected in various tissues, including normal intestinal epithelium (25, 26), the expression of SERCA-type enzymes has not been studied in colon and gastric cancer so far.

The calcium content of the ER lumen is a key determinant controlling apoptosis induced by physiologic stimuli (14, 27). The modulation of the calcium content of the ER by Bcl-2 is involved in the regulation of the apoptotic potential of the cell (14, 28), and the regulation of SERCA expression and function by Bcl-2 is thought to be involved in this process (29). In short term experimental settings, the direct pharmacological inhibition of calcium pumping activity has been shown to lead to growth arrest, differentiation, or caspase-12-dependent apoptosis, depending on the cell type (30-33), and highly specific SERCA inhibitors such as thapsigargin or 2,5-di-tert-butyl-1,4-hydroquinone are known tumor promoters in vivo and in vitro (34, 35) when applied chronically. Moreover, endogenously expressed truncated SERCA variants have recently been implicated in the modulation of the apoptotic potential of the cell by interfering with SERCA-dependent calcium transport (36). In addition, peptide hormone receptors that mobilize calcium from the endoplasmic reticulum have been shown to be involved in positive feedback mechanisms regulating colon cancer cell proliferation and behavior (37, 38). All these data taken together suggest that cellular phenotype, proliferation status, apoptotic potential, and stage of differentiation are intricately connected to ER calcium homeostasis. However, the mechanisms involved in these processes are poorly understood. To better understand the role of SERCA enzymes in epithelial maturation and to shed light on the involvement of the calcium homeostasis of the ER in epithelial malignancies, in this work we investigated the expression of SERCA enzymes in a series of human colon and gastric cancer cell lines, carcinoma tissue, and primary cells, and we studied the modulation of SERCA expression and function during cell differentiation.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells and Treatments-- The Caco-2, SW-48, SW-403, LS-174T, LoVo, SW-620, DLD-1, HT-29 (wild type), and COLO-205 colon cancer cell lines, as well as the KATO-III, NCI-SNU-1, NCI-SNU-16, NCI-N87, and RF-48 gastric cancer lines were purchased from, and cultivated according to the instructions of, ATCC (Manassas, VA), with the modification that RPMI-based media contained Glutamax-I (alanyl-glutamine) in addition to 2 mM glutamine. HT29-5M21 cells were cultured in the presence of 10 µM methothrexate as described previously (39). The culture of cells obtained from crypts of human fetal ileum (HIEC cells) as well as the isolation and culture of primary differentiated ileal epithelial cells have been described earlier (40, 41). Adult primary colonic epithelial cells were obtained as described previously (42).

Exponentially growing cells were trypsinized and seeded into 20-cm2 cell culture dishes at a density of 2 × 104 cells/cm2. When cells reached 80% confluency by microscopic examination (day 2 or 3 post-plating, depending on the rate of growth), medium was renewed, and drugs were added from concentrated stock solutions. Cells that grow in suspension or in a semiadherent manner (KATO-III, NCI-SNU-1, NCI-SNU-16, RF-48) were seeded at an initial density of 2 × 105 cells/ml at the beginning of treatments. Sodium salts of short chain fatty acids and of their analogs were dissolved in phosphate-buffered saline at a concentration of 0.3 M. When only the free acid forms were available commercially (Sigma), these were neutralized by dissolving in 0.3 M sodium bicarbonate at the same concentration and were sterile-filtered. Due to their hydrophobicity, 1,2,3-tributyrylglycerol (tributyrin, Fluka, Germany) and pyvaloyloxymethylbutyrate (Calbiochem) were dispersed to the desired final concentrations as fine emulsions in complete medium by sonication immediately prior experiments. Suberoylanilide hydroxamic acid (SAHA) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), and apicidin was from Calbiochem. Herbimycin A, HC toxin, and thapsigargin were from Sigma. Drugs were added to the cell cultures from concentrated stock solutions made in dimethyl sulfoxide (Me2SO). The final concentration of Me2SO vehicle did not exceed 0.1%, was included in control experiments, and did not interfere with the assays. Untreated control cells were harvested in the exponential phase of non-confluent growth.

After treatments as indicated on figures, the cells were quickly washed twice with ice-cold NaCl (150 mM), precipitated with 5% trichloroacetic acid overnight at 4 °C, and centrifuged (20). The protein pellet was then dissolved in sample buffer (20), and equal amounts of lysates (100 µg of protein per well) were run on 8% SDS-polyacrylamide gels and electroblotted onto nitrocellulose (Hybond ECL, Amersham Biosciences). The transfer of proteins onto nitrocellulose was controlled by Ponceau Red staining. Immunodetection of SERCA proteins using the IID8 (SERCA2-specific) and the PLIM430 (SERCA3-specific) monoclonal antibodies was performed as described previously (20), with the modification that the sample lysis buffer contained also 1 mM phenylmethylsulfonyl fluoride, 0.1 mM aminoethylbenzylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A, freshly added from 1000-fold concentrated stock solutions made in Me2SO.

Rabbit polyclonal antibodies used in this work that specifically recognize the various SERCA3 isoenzymes (SERCA3a, -3b, and -3c) have been characterized previously in detail (24). Immunostaining for the detection of carcinoembryonic antigen and of dipeptidyl peptidase IV was performed using the C6G9 (Sigma) and the HBB 3/775/42 (43) monoclonal antibodies, respectively, at a 1000-fold dilution of the ascites using the electrophoresis and immunostaining system as outlined above. ZO-1 protein was immunostained using an affinity-purified rabbit polyclonal antibody obtained from Zymed Laboratories Inc. that recognizes both the alpha - and alpha + isoforms, after electrophoresis of cellular proteins in 6.5% polyacrylamide gels. The luminescent signal obtained by using the Amersham Biosciences enhanced chemiluminescence system was quantified by scanning non-saturated luminograms (on Kodak Biomax ML films) with an Epson Perfection Photo 1240U scanner using the Adobe Photoshop software (Adobe Systems Inc., Mountain View, CA) and quantitated using the Scion Image software (version 4.0.2, Scion Corp., www.scioncorp.com). Due to the absence of detectable SERCA3 in untreated KATO-III or Caco-2 cells, SERCA expression in these cells was expressed on figures in percentages, with end point signal being taken arbitrarily as 100%.

Primary Cells-- Primary colon cancer cells were obtained from fresh surgical specimens. Homogeneous tumor tissue was carefully separated from adjacent structures, cut into sub-millimeter sized pieces with a scalpel, and placed into 24-well plates in a medium consisting of a mixture of equal volumes of RPMI 1640 and Ham's F-12 nutrient medium supplemented with glutamax-I, glutamine, sodium pyruvate, nonessential amino acids, vitamins, and reduced glutathione (all reagents obtained from Invitrogen) plus 20% decomplemented fetal calf serum, 50 units/ml penicillin, 50 µg/ml streptomycin, and 2.5 µg/ml amphotericin B. After 3 days in culture, floating tissue debris was aspirated, and growth of adherent cells and morphology, as well as the absence of fibroblasts or microbial contamination, were monitored microscopically. Only cultures devoid of fibroblast contamination were used. CEA synthesis was detected by immunoblotting of total cell lysates as indicated above. The cells were first treated with short chain fatty acids at day 6 post-plating in the wells where they had been originally plated and then after 4 months of continuous growth and regular subculturing in 20-cm2 Petri dishes.

Immunohistochemistry-- Staining of 5-µm thick cryostat sections of freshly frozen normal and malignant colon and stomach tissue with the SERCA3-specific PLIM430 monoclonal antibody was performed as follows. Slides were allowed to dry overnight at room temperature. The sections were then fixed in acetone for 10 min at room temperature, were allowed to dry, and were rehydrated in Tris-buffered saline (TBS, pH 7.4) containing 0.1% Tween 20 (TBS/Tween) for 10 min. Inhibition of nonspecific protein binding was performed by incubation for 30 min in TBS/Tween supplemented with 5% nonfat dry milk. The PLIM430 antibody, purified previously by protein A affinity chromatography, was then applied upon the sections in the above solution at 1 µg/ml concentration and incubated at room temperature for 90 min. The slides were then rinsed with distilled water three times and incubated in TBS/Tween/milk for 10 min. After repeating this washing step, the slides were rinsed three times with TBS/Tween and incubated for 10 min in TBS containing 1/30 volume normal horse serum. Slides were incubated with biotinylated anti-mouse secondary antibody (Vectastain ABC kit, Vector Laboratories) for 1 h, followed by incubation with avidin-biotin-peroxidase complex (Vectastain ABC kit) for 45 min according to the protocol of the manufacturer. As chromogen 3,3'-diaminobenzidine was used, the slides were counterstained with hematoxylin. In control experiments the primary antibody was omitted or replaced by an isotype-matched irrelevant antibody, and this resulted in no staining.

Calcium Fluorimetry-- Fluorescence from suspensions of Fura-2-loaded KATO-III cells was recorded at 37 °C with a Shimadzu RF-1501 spectrofluorimeter (Shimadzu Europe, Duisburg, Germany) equipped with a stirring apparatus. For calculation of cytosolic calcium concentrations, the ratio of fluorescence at excitation wavelengths of 340 and 380 nm (emission at 510 nm) was calibrated according to Grynkievicz (44). 13 × 106 KATO-III cells were loaded with 1.7 µM Fura-2-AM (Sigma) in 6 ml of complete culture medium for 45 min at 37 °C. The cells were then centrifuged for 8 min at 350 × g and resuspended in 10 ml of fresh complete medium. After 15 min at 37 °C the cells were washed by centrifugation with 10 ml of buffer containing 136 mM NaCl, 2.7 mM KCl, 10 mM Hepes, pH 7.45, adjusted with NaOH, 2 mM MgCl2, 1 mg/ml D-glucose, and 1 mM CaCl2 and resuspended in 14 ml of this buffer. Measurements were performed with cuvettes containing 2 ml of cell suspension. To measure SERCA-dependent intracellular calcium storage capacity, calcium mobilization from the ER into the cytosol was induced with 1 µM thapsigargin in the presence of 2 mM extracellular EGTA, and peak cytosolic calcium concentration was recorded.

RT-PCR Amplification of SERCA Transcripts-- Total cellular RNA was isolated from cells using the Trizol reagent (Invitrogen) according to the instructions of the manufacturer. 500 ng of total RNA was reverse-transcribed using the murine leukemia virus-reverse transcriptase (PerkinElmer Life Sciences). After inactivation of the reverse transcriptase, semiquantitative PCR was initiated by adding 0.625 units of AmpliTaq Gold DNA polymerase (PerkinElmer Life Sciences) in a 25-µl reaction mixture. Touchdown PCR (45) was performed for 10 cycles with an annealing temperature decrement from 65 to 56 °C in order to increase the specificity of priming during initial cycles of amplification. PCR was then conducted essentially as described (45) with slight modifications as follows. 18 cycles of PCR were conducted for SERCA2b, 22 cycles for SERCA3a, 23 cycles for -3b, and 24 cycles for -3c, with each cycle consisting of successive periods of 1 min at 95 °C, 1 min at 58 °C, and 1 min at 72 °C with a final extension step of 10 min at 72 °C. The SERCA isoform-specific oligonucleotide primers used are as follows: SERCA2-5', 2861TCA TCT TCC AGA TCA CAC CGC T2882; SERCA2b-3', 3129TCA AGA CCA GAA CAT ATC GC3110; SERCA3-5' (same for the various isoforms), 2674GAG TCA CGC TTC CCC ACC ACC2694; SERCA3a-3', 2992GGC TCA TTT CTT CGT GCA TGT GGT TC2967; SERCA3b-3', 3080GGC TCA TTT CTT CCG GTG TGG TC3058; and SERCA3c-3', 3093GGC TCA TTT CTT CAA AGA GGC CAA C3069. As internal control glyceraldehyde-3-phosphate dehydrogenase mRNA was amplified as described previously (45). The amplification products were separated in 1.5% agarose gels and visualized by ethidium bromide staining. The apparent molecular masses of the PCR products corresponded to those calculated based on the reported sequences; moreover, the identity of the PCR products was also confirmed by direct sequencing (Genome Express, Grenoble, France). Quantitative data were obtained using the Scion Image software (see above) on digitized images of ethidium bromide-stained gels. Data in this work correspond to at least three independent experiments and are presented as means ± S.E.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SERCA3 Expression Is Lost in Colon Carcinomas-- In order to study SERCA3 expression in human tissue, we developed an immunohistochemical staining protocol using the SERCA3-specific PLIM430 monoclonal antibody. As illustrated in Fig. 1, panel A, SERCA3 was readily detected in normal colonic crypt epithelium in enterocytic as well as mucus-secreting cells and in stomach mucosa (panel B). In colonic crypts stronger SERCA3 staining could be seen in more mature cells residing in the luminal region. However, SERCA3 could also be detected in deeply located, less mature cells, suggesting that SERCA3 expression is induced early in colonic epithelial differentiation. On the other hand, when SERCA3 expression was investigated in colon cancer tissue, in 9 cases of the 12 examined, a complete lack of staining in the malignant cells was observed (panels C-G), and in the remaining cases a faint staining could be observed in the apical region of the cells (panel H). At the same time, adjacent normal epithelial tissue stained strongly positive for SERCA3 in all specimens in a highly reproducible manner. These data show that SERCA3 expression is dramatically decreased or completely lost in colon adenocarcinomas, although present at high levels in normal epithelium.


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  		Fig. 1.   SERCA3 expression in normal colonic and gastric mucosa and in colonic neoplasia. Cryostat sections were labeled with the SERCA3-specific PLIM430 antibody using an avidin-biotin-peroxidase system and are shown as a brown coloration with diaminobenzidine on a blue hematoxylin counterstain. Panel A, normal colonic epithelium, longitudinal section. Panel B, gastric mucosa, longitudinal section. Panel C, moderately differentiated adenocarcinoma (SERCA3neg) surrounded by normal epithelial crypts (SERCA3pos; cross-sectioned). Arrowheads delineate the interface between normal (brown coloration; SERCA3pos) and cancerous tissue (blue coloration; SERCA3neg). Panel D, higher magnification view of a region of the specimen presented in panel C, displaying a normal (SERCA3pos) and an adenocarcinomatous gland (SERCA3neg). Panels E-G, colon adenocarcinoma specimens (SERCA3neg) and adjacent normal epithelium (SERCA3pos). Panel F, arrowheads delineate the interface between normal (brown coloration; SERCA3pos) and cancerous tissue (blue coloration; SERCA3neg). Panel H, colon adenocarcinoma displaying weak apical SERCA3 staining with adjacent normal epithelium. Inset, higher magnification view of malignant cells with weak apical staining for SERCA3. Panels I and J, immunocytochemical staining for SERCA3 in KATO-III (SERCA3neg) and COLO-205 (SERCA3pos) cells, respectively. A moderate SERCA3 staining can be observed in COLO-205 cells.

In accordance with immunoblotting data presented in Fig. 2, SERCA3 expression could also be detected by immunocytochemistry in the COLO-205 cell line (panel J), whereas the KATO-III cell line was negative (panel I). SERCA3 expression in COLO-205 cells was, however, markedly weaker than that observed in normal colonic epithelial cells.


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  		Fig. 2.   Expression of SERCA-type calcium pumps in colon and gastric cancer cell lines. Equal amounts of total cellular protein from various exponentially growing colon (panel A) and gastric (panel B) cancer cell lines and from normal adult colonic epithelial cells (panel C) were immunostained for SERCA2 and SERCA3 expression with the IID8 and PLIM430 antibodies, respectively. Although SERCA2 expression is relatively homogeneous, SERCA3 expression varies among cell lines. In all cell lines, however, SERCA3 expression is inferior to that of normal colonic epithelial cells.

SERCA3 Expression in Colon and Gastric Cancer Cell Lines-- In order to study SERCA3 expression in various colon and gastric cancer cell lines, cells in the exponential phase of growth were harvested, and total cellular protein was probed with the PLIM430 (pan-SERCA3 specific) monoclonal antibody in a Western blot format as described previously (20), using a very sensitive chemiluminescent detection method. As an internal control, SERCA2 was detected using the IID8 monoclonal antibody as well. As shown in Fig. 2, although SERCA2 was expressed in all cell types at comparable levels, the expression of SERCA3 was undetectable in DLD-1, Caco-2, KATO-III, or NCI-SNU-1 cells. Although SERCA3 could be detected at various levels in HT29, SW-403, SW-48, LS-174T, COLO-205, NCI-N87, and NCI-SNU-16 cells, freshly isolated normal adult colonic epithelial cells expressed SERCA3 protein at a higher level than any of the cell lines (Fig. 2). SERCA3 protein was also detected in primary differentiated embryonic ileal epithelial cells and in cells obtained from crypts of human fetal ileum (HIEC cells, not shown).

SERCA3 Expression Increases during Drug-induced Cell Differentiation-- As blockage of differentiation is a general hallmark of malignancy, to investigate the implication of SERCA3 in colon cancer differentiation, cell lines were treated with sodium butyrate, a known physiological differentiation-inducing agent of colonic mucosa, and SERCA expression was investigated. As shown in Fig. 3, panel a, butyrate induced SERCA3 expression in various colon and gastric cancer cell lines in a concentration-dependent manner in the low millimolar range. At the same time, SERCA2 expression did not change or decreased. Induction of SERCA3 by butyrate occurred for 2-5 days (Fig. 3, panel b) with maintained viability. Similar results were also obtained in the LoVo, SW-620, and RF-48 cell lines (not shown). In accordance with data in the literature (46-48), butyrate treatment was accompanied by growth arrest, and longer treatments at higher concentrations led to apoptosis.


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  		Fig. 3.   Induction of SERCA3 expression by butyrate in various colon and gastric cancer cell lines. Panel a, cells were cultured in the presence of various concentrations of sodium butyrate for 4 days, and the expression of SERCA2 and SERCA3 enzymes was determined by Western blotting. Butyrate treatment induced the expression of SERCA3 in a concentration-dependent manner in the low millimolar range. Induction was manifest in cells with undetectable initial SERCA3 levels (KATO-III and DLD-1) and in cells that express this enzyme already in the untreated state (LS-174T, SW-48, SW-403, and NCI-N87). Panel b, time course of SERCA3 induction by butyrate. Cells were cultured in the presence of 3 mM butyrate, and SERCA expression was measured daily. Induction of SERCA3 expression is detectable as early as day 1 and reaches a plateau after 4-5 days of treatment in most cell lines.

Based on their negligible basal expression of SERCA3 and strong induction upon butyrate treatment, KATO-III cells were selected for further quantitative study. As illustrated in Fig. 4, induction of SERCA3 expression and down-regulation of SERCA2 expression were manifested at low millimolar butyrate concentrations (panel A), starting at day 1 following treatment (panel B). Under these conditions butyrate treatment induced growth arrest (panel D) with maintained cell viability (panel C). Similar results were obtained on DLD-1 cells as well (not shown).


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  		Fig. 4.   Quantitative evaluation of expression of SERCA proteins in butyrate-treated KATO-III cells. Panel A, concentration dependence of SERCA expression. The cells were treated with various concentrations of butyrate for 4 days, and SERCA expression was determined. Filled circles, SERCA3; open circles, SERCA2. Panel B, time course of SERCA expression. KATO-III cells were treated with 3 mM butyrate, and SERCA expression was determined daily for 4 days. Filled circles, SERCA3; open circles, SERCA2. Panel C, viability of butyrate treated KATO-III cells. The cells were treated with 3 mM butyrate for 4 days, and viability was determined by trypan blue exclusion daily. Filled circles, butyrate-treated cells; open circles, untreated control cells. Panel D, inhibition of proliferation of KATO-III cells by butyrate. The cells were treated with 3 mM butyrate for 4 days, and cell density was measured daily by a hemocytometer. Filled circles, butyrate-treated cells; open circles, untreated cells.

Analysis of the modulation of SERCA expression upon butyrate treatment of single cell clones, obtained by limiting dilution cloning, showed that the cell line behaved in a clonally homogeneous manner. In all KATO-III clones tested, butyrate treatment strongly and homogeneously induced SERCA3 expression, whereas SERCA2 expression was at the same time decreased (Fig. 5). Similar results were obtained with single cell clones of the DLD-1 colon cancer cell line as well (not shown).


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  		Fig. 5.   Induction of SERCA3 expression in single cell clones of KATO-III. The expression of SERCA2 and SERCA3 enzymes of single cell clones obtained from the KATO-III cell line was compared prior to and after treatment with 3 mM butyrate for 4 days. Induction of SERCA3 expression was obtained in all KATO-III clones (12 clones tested).

As shown in Fig. 6, panel A, other short chain fatty acids such as propionate, valerate, isobutyrate, isovalerate, caproate, and valproate also induced SERCA3 expression, as well as the butyrate-generating prodrugs tributyrin (tributyrylglycerol) and pivaloyloxymethyl butyrate (49-51). Other histone deacetylase inhibitors such as HC toxin, apicidin, and SAHA also induced SERCA3 expression in accordance with the literature (52-54) and to a lesser extent than butyrate. Conversely, and similar to previous data, acetate and other chemicals structurally related to butyrate, such as crotonate, cyclopropane-carboxylate, pentenoate, pentinoate, trimethylacetate, were only marginally active or had no effect, and heptafluorobutyrate, pivaloate, gamma -aminobutyrate, or lactate were inactive (not shown). Aryl derivatives such as 3-phenylpropionate and 4-phenylbutyrate also induced SERCA3 expression, whereas trans-cinnamate or phenoxyacetate were without effect (not shown). These observations are in agreement with previous data in the literature regarding the pharmacological profile of these molecules (55, 56), and with the recently established three-dimensional tube-like pocket structure of the active site of histone-deacetylase where inhibitors bind (57).


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  		Fig. 6.   Panel A, induction of SERCA3 expression by butyrate analogs. KATO-III cells were treated as follows: with various naturally occurring short chain fatty acids (acetate, propionate, butyrate, valerate, and caproate, all at 3 mM); with butyrate-releasing prodrugs (1 mM tributyrin and 0.5 mM pivaloyloxy-methylbutyrate); with synthetic analogs containing double and triple bonds (crotonate, vinylacetate, pentenoic, and pentinoic acid), a branching chain (isobutyrate, isovalerate, trimethylacetate, and valproate), a cycloalkyl group (cyclopropane-carboxylic acid) or an aryl group (phenylacetate, phenylpropionate, and phenylbutyrate) at 3 mM concentration for 5 days; and with the highly specific histone deacetylase inhibitors HC toxin (0.1 µM), SAHA acid (1 µM), or apicidin (1 µM). Short chain fatty acids, their prodrugs, as well as aryl-substituted analogs and various histone deacetylase inhibitors induced SERCA3 expression. Panel B, growth inhibition is not required for SERCA3 induction. KATO-III cells were incubated with 3 mM butyrate, 3 mM valerate, or cultured without drugs in serum-free medium containing 0.5% bovine serum albumin (BSA). Cell densities (lower panel) and SERCA3 expression (upper panel) were detected at day 3. Valerate treatment induced SERCA3 expression without growth arrest, and growth inhibition induced by serum withdrawal was without effect.

Interestingly, as shown in Fig. 6, panel B, although as potent as butyrate in terms of SERCA3 induction, valerate treatment did not induce growth arrest in KATO-III cells under the conditions used. Additionally, when the cells were cultured without drugs in medium in which serum was replaced by 0.5% bovine serum albumin leading to a significant inhibition of proliferation, no SERCA3 expression was seen. These observations suggest that growth inhibition per se is not indispensable for induction of SERCA3 expression by short chain fatty acids.

Short chain fatty acids induced SERCA3 expression in primary cultures of colon cancer cells as well. As shown in Fig. 7, left panel, SERCA3 expression of primary cells was increased approximately 7-9-fold by a 5-day treatment with 3 mM butyrate or valerate, whereas the expression of SERCA2 at the same time diminished. Essentially the same effect could be observed in cells, which had been grown continuously for 4 months in vitro (right panel) before treatments.


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  		Fig. 7.   Induction of SERCA3 expression in freshly isolated primary colon cancer cells. Cells at an early stage following plating (day 6, left) and after 4 months of continuous growth and subculturing (right) were treated with 3 mM butyrate or valerate for 5 days. In both cases a strong induction of SERCA3 expression was obtained by both SCFA.

In addition to butyrate and its analogs, the effect of structurally and pharmacologically unrelated molecules, such as the tyrosine kinase inhibitor herbimycin A and suramine which are drugs that have been reported to induce the differentiation of COLO-205 and NCI-SNU-16 cells, respectively (58, 59), has been studied. As shown in Fig. 8, panels A and B, both drugs induced SERCA3 expression in their target cells, indicating that this effect is not restricted only to short chain fatty acids or other histone deacetylase inhibitors.


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  		Fig. 8.   Induction of SERCA3 expression by various differentiation inducing agents. Panel A, COLO-205 cells were treated with 0.52 µM herbimycin A, and the time course of the expression of SERCA2 and SERCA3 proteins was determined. Panel B, NCI-SNU-16 cells were treated with various concentrations of suramin for 7 days, and SERCA2 and SERCA3 expression was measured. Treatment of the cells with these differentiation-inducing agents led to the induction of the expression of SERCA3 protein.

SERCA Expression during Differentiation of Caco-2 and HT29-5M21 Cells-- Caco-2 cells, a human colon adenocarcinoma cell line, spontaneously undergo differentiation in post-confluent cultures. The initially rapidly growing cells become quiescent and display structural, biochemical, and functional characteristics corresponding to a mature enterocytic phenotype (60). After reaching confluency the cells stop to proliferate, elaborate tight junctions, microvilli, transcellular solute transport, display transepithelial electric resistance, and express many differentiation markers such as carcinoembryonic antigen (CEA), sucrase-isomaltase, dipeptidyl peptidase IV, alkaline phosphatase, and others. Due to their differentiation potential, Caco-2 cells constitute a widely used model of enterocytic differentiation and function.

When SERCA expression of post-confluent Caco-2 was analyzed, a marked induction of SERCA3 expression was seen (Fig. 9, panel A). Although the expression of SERCA3 was undetectable in exponentially growing non-confluent and early post-confluent cultures, SERCA3 expression was manifest from day 5 to 6 post-confluency and reached a plateau at day 20, whereas SERCA2 expression was only slightly increased. The induction of SERCA3 expression followed a time course very similar to that of carcinoembryonic antigen, a widely used marker of differentiation of this cell line (61). In addition, during this process the expression of dipeptidyl peptidase IV, another marker of differentiation (62), was induced, and the isoform switch from alpha - to alpha + of the tight junction protein ZO-1 was seen as described earlier (63, 64).


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  		Fig. 9.   Differentiation of Caco-2 and HT29-5M21 cells. Panel A, Caco-2 cells were allowed to reach confluency and were cultured for 26 days. Cells were harvested at different time points, and the expression of SERCA2 and SERCA3 proteins was measured. The expression of various established markers of enterocytic differentiation was determined in parallel. Whereas SERCA2 was constitutively expressed in the cells, and its expression slightly increased during the differentiation of post-confluent cells, SERCA3 was undetectable prior confluency and was strongly induced during post-confluent cell differentiation. The time course of SERCA3 expression closely paralleled that of the induction of the expression of carcinoembryonic antigen or dipeptidyl peptidase IV (DPP-IV), as well as that of the isoform switch from alpha - to alpha + of the tight junction associated protein ZO-1. Panel B, induction of SERCA3 expression in HT29-5M21 cells. During growth of the cells in post-confluent conditions that allow differentiation of cells toward a mucus-secreting phenotype, induction of SERCA3 expression was observed.

SERCA expression was also investigated in HT29-5M21 cells, a well characterized methothrexate-resistant clone of HT29. Although non-differentiated in the exponential phase of growth, in post-confluent culture these cells display a well differentiated, goblet cell-like and mucus-secreting phenotype (39). As shown in Fig. 9, panel B, SERCA3 expression was induced during the differentiation of post-confluent HT29-5M21 cells.

SERCA3 Isoenzymes-- Recent data in the literature, including ours, indicate that the SERCA3 gene can give rise by alternative splicing in the 3' region of the primary transcript to three mRNA isoforms, SERCA3a, -3b, and -3c, coding for proteins that carry unique peptide sequences in their C-terminal region (22-24). To investigate SERCA3 expression on the isoform level, we performed semiquantitative RT-PCR experiments using oligonucleotide primers that allow the specific amplification of the various SERCA3 transcripts (45), and we studied the expression of the corresponding protein isoforms using recently developed antipeptide antibodies (24) that recognize unique peptide sequences in SERCA3a, -3b, and -3c, respectively.

As shown in Fig. 10, panel A, normal primary adult intestinal epithelial cells strongly expressed all three SERCA3 isoforms, and the expression of these isoenzymes was significantly induced early during the differentiation of post-confluent Caco-2 cells on the mRNA level (panels A and B). The corresponding protein isoforms could also be detected with isoform-specific antibodies in differentiating Caco-2 cells (panel C), as well as in butyrate-treated KATO-III cells. However, in KATO-III SERCA3a expression was predominant (not shown). The kinetics of the induction of SERCA3 mRNA was somewhat dissimilar when compared with that of the corresponding protein in Caco-2 cells. Induction of SERCA3 mRNA could be detected by RT-PCR as early as day 1 post-confluency for all three isoforms (Fig. 10, panel A) and increased severalfold during early post-confluency (Fig. 10, panel B), after which mRNA levels decreased again (not shown). On the other hand, protein induction could be detected by Western blotting from day 5 post-confluency, and protein expression levels followed thereafter a plateau-type time course. The markedly earlier detection of SERCA3 mRNA is probably due to the higher sensitivity of RT-PCR when compared with that of Western blotting. In addition, as post-confluent differentiation of Caco-2 cells is accompanied by growth arrest, the plateau-type kinetics of the accumulation of SERCA3 protein is compatible with transitory induction of corresponding mRNA, assuming that SERCA3 protein is more stable in these cells than the corresponding mRNA.


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  		Fig. 10.   Expression of SERCA3 isoenzymes in differentiating Caco-2 and normal adult colonic epithelial cells. Panel A, induction of the synthesis of SERCA3a, -3b, and -3c mRNA in Caco-2 cells after confluency as detected by SERCA3 isoform-specific RT-PCR. As control glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as well as SERCA2b were amplified in the same RNA preparations. The various SERCA3 isoforms were abundantly expressed in normal adult human colonic epithelial cells. Panel B, estimation of SERCA isoform mRNA levels in differentiating Caco-2 cells by semiquantitative RT-PCR using isoform-specific primers. As internal controls SERCA2b and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were used. Differentiation of Caco-2 cells leads to the induction of the expression of all three SERCA3 isoforms. Panel C, induction of the expression of SERCA3a, -3b, and -3c protein in post-confluent Caco-2 cells detected by Western blotting using isoform-specific antibodies. Induction of SERCA3a, -3b, and -3c protein expression could be detected during differentiation.

Calcium Homeostasis of Differentiating KATO-III Cells-- In order to study cellular calcium homeostasis in functional terms during drug-induced cell differentiation, untreated as well as butyrate-treated KATO-III cells were loaded with Fura-2 and analyzed by calcium spectrofluorimetry. Resting cytosolic calcium levels and calcium mobilization from the ER into the cytosol upon complete inhibition of cellular SERCA activity using supramaximal concentrations of the specific SERCA inhibitor thapsigargin (32, 65) were quantified. As shown in Fig. 11, the resting cytosolic calcium concentration of butyrate-treated cells was significantly higher than that of controls (panel A), whereas the amount of calcium released from the ER into the cytosol upon SERCA inhibition by thapsigargin (panel B) was decreased in butyrate-treated cells, indicating decreased calcium storage in the ER. These data show that the calcium homeostasis of the KATO-III cell line undergoes a significant remodeling during drug-induced differentiation.


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  		Fig. 11.   Calcium homeostasis of KATO-III cells differentiated by butyrate. KATO-III cells were treated for 5 days with 3 mM butyrate, and cytosolic calcium concentration [Ca2+]i was compared with untreated controls in resting cells (panel A) and following treatment with a supramaximal dose of the SERCA inhibitor thapsigargin (1 µM, panel B). Resting cytosolic calcium concentration was significantly higher in butyrate-treated cells than in controls, and the amount of calcium stored in intracellular pools that could be released into the cytosol by thapsigargin was significantly decreased in butyrate-treated cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

As shown in this work, SERCA3 expression is strongly decreased or completely lost in colon carcinomas while being abundantly expressed in normal colonic and gastric epithelial cells. Similarly, in several colon and gastric cancer cell lines this enzyme was absent, and in others, in which SERCA3 expression could be detected by a highly sensitive chemiluminescent immunoblotting method, this was significantly inferior to that observed in purified primary normal colonic epithelial cells. At the same time, the ubiquitous SERCA2b isoform was present at similar levels in all studied cell types.

Independently from the initial level of SERCA3 expression, the differentiation of cell lines of enterocytic (such as Caco-2) as well as of mucus-secreting phenotypes (HT29-5M21) was accompanied by a marked increase of the expression of this enzyme. SERCA3 expression could be induced by several physiologically relevant short chain fatty acids such as butyrate, valerate, and to a lesser extent by propionate or caproate, by synthetic butyrate analogs such as phenylbutyrate, phenylpropionate, or phenylacetate (57), or butyrate-releasing prodrugs such as pivaloyloxymethyl-butyrate (66) or tributyrin (50), molecules with clinical potential for differentiation induction therapy of malignancies (49, 50).

SERCA3 expression was also obtained with drugs such as the tyrosine kinase inhibitor herbimycin A or by suramin, known to induce the differentiation of the cell lines studied (58, 59). In addition, a strong induction of SERCA3 expression, including all three isoforms, was also observed during the spontaneous differentiation of post-confluent Caco-2 cells, with a time course comparable with that of other established markers of differentiation, such as carcinoembryonic antigen (61) or dipeptidyl peptidase IV expression (62), or the shift from the alpha - to the alpha + isoform of the ZO-1 protein (63, 64).

To study the effect of short chain fatty acids on primary colon cancer cells, we attempted to establish short term cultures as well as continuously growing cell lines from tumor tissue from freshly obtained surgical specimens. Despite the difficulties inherent to this technique due to microbial contamination of the specimens and poor plating efficiency and growth of the primary tumor cells in vitro, out of 12 attempts we successfully established primary cultures as well as a permanently growing, CEA-expressing cell line from a moderately differentiated mucus-secreting primary adenocarcinoma of the descending colon of a 61-year-old man. Cells in short term culture behaved very similarly to other established cell lines in terms of SERCA3 induction by SCFA, and SERCA3 expression remained inducible even after 4 months of continuous growth and subculturing. Although the detailed characterization of the obtained cell line requires more detailed study and despite potential heterogeneity of the primary cultures, this observation suggests that short chain fatty acids can induce SERCA3 expression in primary colon cancer cells similarly to that seen in permanent cell lines. Therefore, studies performed on established cell lines constitute a physiologically relevant approach in this regard.

Short chain fatty acids are produced by the fermentation of dietary fibers by the colonic flora, and the induction of the differentiation followed by apoptosis of microscopic precancerous lesions by these molecules (and in particular by butyrate) is considered as being a main mechanism of the protective effect of a fiber-rich diet against colorectal cancer (67, 68). From these data it is tempting to speculate that cellular calcium homeostasis may be modulated by short chain fatty acids in the colonic epithelium. In addition, our data suggest that this effect may also operate in the case of gastric cancer as well.

Inhibition of histone deacetylases is a key component of the mechanism of action of differentiation induction by butyrate (69). Highly specific histone deacetylase inhibitors such as HC toxin, apicidin, or SAHA also induced SERCA expression in our hands, indicating that this mechanism may be involved in the modulation of SERCA3 expression by short chain fatty acids. Butyrate appeared, however, to be a more potent inducer of SERCA3 expression. This is in accordance with data in the literature obtained in various experimental systems (52-54) and may be due to differences in stability of the drugs in cell culture conditions, and in addition to histone-deacetylase inhibition, butyrate and other SCFAs also interact with other intracellular targets as well (70).

The functional implications of the modulation of SERCA expression during epithelial differentiation are complex. The pattern of a calcium signal is shaped by the concerted and coordinated action of mechanisms that increase cytosolic calcium levels (i.e. calcium channels) and that decrease it (calcium pumps), leading in many instances to oscillatory calcium signals. SERCA enzymes are actively resequestering calcium into the endoplasmic reticulum, even during calcium mobilization from this organelle, and thus clearly contribute to the shaping of calcium transients (71, 72). The various geometrical characteristics of a calcium transient and the frequency and amplitude of repetitive calcium oscillations convey key information to calcium-activated intracellular targets. The modulation by the cell of the spatiotemporal characteristics of calcium oscillations confers specificity and selectivity to calcium signals, because calcium-activated target molecules such as calmodulin-dependent protein kinases, calcineurin, or protein kinase C isoenzymes are optimally activated at distinct frequencies and amplitudes of calcium oscillations (73-75). This can lead to differential activation of transcription factors such as NF-AT, NF-kappa B, and others, leading to the modulation of gene expression by calcium oscillations (76-78). The biochemical characteristics of SERCA2b and of SERCA3 enzymes are distinct (79). In particular, the calcium affinity of SERCA3 is lower (KCa = 1.2 µM) than that of SERCA2b (KCa = 0.2 µM). Quantitative changes of the relative abundance of various SERCA isoenzymes thus may alter resting cytosolic calcium levels and may modify the shape of a calcium transient and of calcium oscillations (80), leading to an altered responsiveness of the cell to stimuli. For example if calcium elimination from the cytosol by SERCA enzymes is modified due to the replacement of SERCA2b by SERCA3, a quantitatively identical IP3 generation may lead to modified calcium transients. Data presented in this work showing that resting cytosolic calcium levels are higher and calcium mobilization is smaller in butyrate-treated KATO-III cells, where SERCA2b expression is decreased and SERCA3 expression is increased, are compatible with these observations. Modulation of SERCA expression may thus contribute to the fine-tuning of calcium signaling and may reset and modify activation thresholds and thus may contribute to redirect calcium signals toward distinct intracellular target proteins and signaling pathways.

Moreover, as the endoplasmic reticulum consists of heterogeneous sub-compartments within a single cell in terms of calcium content, relative abundance of IP3 receptors, and SERCA enzymes (81-86), modulation of SERCA expression may also reflect the remodeling of structurally as well as functionally distinct sub-compartments of the organelle. The present work indicates that colonic epithelial cells may constitute an interesting model system for the study of ER heterogeneity.

The inability to follow a complete normal differentiation program is a common hallmark of most malignant cells. Although the degree of malignancy and the state of differentiation of colon cancers may vary somewhat independently, cancerous cells almost always present defects in the expression of genes associated with a fully mature normal phenotype. The data presented in this work show for the first time that SERCA3 expression is deficient in colon cancers and that differentiation of these cells leads to the induction of its expression. On the other hand, in normal epithelium, although more strongly expressed in more luminally located cells, SERCA3 expression could be detected already in less mature cells, residing in deeper regions of crypts, suggesting that SERCA3 expression may be a relatively early event during normal differentiation.

An in depth understanding of the complex structural and functional implications of the reorganization of the endoplasmic reticulum during differentiation will require the simultaneous analysis of the expression of many genes as in Ref. 87. In particular, due to the extreme complexity and plasticity of the different calcium regulatory systems that are interconnected and work in concert to handle cellular calcium fluxes, it is not possible at the present time to associate growth control and cellular differentiation to one particular calcium pump species. An in depth understanding of the implications of the modulation of SERCA expression on cellular calcium homeostasis and differentiation will require further investigation on several cell types, using inducible expression vectors coding for SERCA isoenzymes as well as isoform-specific gene knock out techniques. Our work shows, for the first time, that induction of SERCA3 expression is taking place during the differentiation of colon cancer cells, and data obtained on the KATO-III cell line suggest that cellular calcium homeostasis of the ER may also be reorganized in functional terms during differentiation. In addition, our data indicate that SERCA3 may serve as a useful new marker for the study of colon as well as gastric cancer phenotypes.

    ACKNOWLEDGEMENTS

We thank Dr. A. Habib (Unité 348 INSERM, Paris, France) for giving us the DLD-1 and the HT29 (wild type) colon cancer cell lines and Prof. N. Crawford (Royal College of Surgeons of England, London, UK) for the PLIM430 hybridoma. The useful discussions we had with Dr. A. Enyedi and Dr. B. Sarkadi (National Institute of Haematology and Immunology, Budapest, Hungary), with Dr. C. Chomienne (Hôpital SaintLouis, Paris, France), Prof. J. Chambaz, Dr. T. Lesuffleur (Unité 505 INSERM, Paris, France), Dr. C. Gespach (Unité 482 INSERM, Paris, France), Dr. I. Sobhani (Unité 10 INSERM, Paris, France), and Evelyne Dupuy (Unité 348 INSERM, Paris, France) are greatly appreciated. We thank Dr. S. Lévy-Tolédano (Unité 348 INSERM, Paris, France), Prof. J. Mikol (Service d'Anatomie Pathologique, Hôpital Lariboisière, Paris, France), and Prof. P. Valleur (Service de Chirurgie Générale et Digestive, Hôpital Lariboisière, Paris, France) for supporting this work, and K. Walther (University of Regensburg, Germany) for expert technical assistance with the isolation of primary epithelial cells.

    FOOTNOTES

* This work was supported in part by INSERM, by the Association pour la Recherche sur le Cancer, France, by Nestlé, France, by Research Grant T 032766 from OTKA, Hungary, and by APRIFEL, France.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. Section 1734 solely to indicate this fact.

We dedicate our work to the memory of all victims of the tragedy of September 11, 2001.

§ Recipient of a doctoral fellowship from Nestlé, France, and from APRIFEL.

|| Recipient of the Bolyai János Research Fellowship of the Hungarian Academy of Sciences.

|||| To whom correspondence should be addressed: U. 348 INSERM, Hôpital Lariboisière, 8 Rue Guy Patin, 75010 Paris, France. Fax: 33-1- 49-95-85-79; E-mail: bela.papp@inserm.lrb.ap-hop-paris.fr.

Published, JBC Papers in Press, May 1, 2002, DOI 10.1074/jbc.M201747200

    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; SERCA, sarco/endoplasmic reticulum calcium ATPase; IP3, myo-inositol-1,4,5-trisphosphate; SCFA, short chain fatty acids; CEA, carcinoembryonic antigen; ZO-1, zonula occludens protein-1; Me2SO, dimethyl sulfoxide; RT, reverse transcriptase; SAHA, suberoylanilide hydroxamic acid; TBS, Tris-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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