The NF-jB target genes ICAM-1 and VCAM-1 are differentially regulated during spontaneous differentiation of Caco-2 cells

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1 The NF-jB target genes ICAM-1 and VCAM-1 are differentially regulated during spontaneous differentiation of Caco-2 cells Erhan Astarci 1, Asli Sade 2, Ismail Çimen 2, Berna Savas 3 and Sreeparna Banerjee 1,2 1 Department of Biochemistry, Middle East Technical University, Ankara, Turkey 2 Department of Biological Sciences, Middle East Technical University, Ankara, Turkey 3 Department of Pathology, Ankara University School of Medicine, Sihhiye, Ankara, Turkey Keywords Caco-2; C EBPb; differentiation; NF-jB; PKC Correspondence S. Banerjee, Department of Biological Sciences, Z-16, Middle East Technical University, Ankara 06800, Turkey Fax: Tel: banerjee@metu.edu.tr (Received 15 December 2010, revised 19 June 2012, accepted 20 June 2012) doi: /j x Intestinal epithelial differentiation entails the formation of highly specialized cells with specific absorptive, secretory, digestive and immune functions. Cell cell and cell microenvironment interactions appear to be crucial in determining the outcome of the differentiation process. Using the Caco-2 cell line, which undergoes spontaneous re-differentiation when grown past confluency, we observed a loss of VCAM-1 (vascular cell adhesion molecule 1) mrna expression, while ICAM-1 (intercellular cell adhesion molecule 1) mrna expression was seen to increase over the course of differentiation. Protein kinase Ch (PKCh) acted downstream of protein kinase Ca (PKCa) to inactivate inhibitor of jb (IjB) and activate nuclear factor jb (NF-jB) in undifferentiated cells, and this pathway was inhibited in the differentiated cells. The increase in ICAM-1 mrna expression in the differentiated cells was due to increased promoter recruitment of C EBPb, which transcriptionally up-regulated ICAM-1 mrna. However, protein expression of ICAM-1 was found to decrease over the course of differentiation due to degradation in the proteasome and lysosome. Immunohistochemistry using tumor samples from colon cancer patients indicated that non-transformed matched normal cells (well-differentiatied) showed no ICAM-1 expression, but the poorly differentiated tumor cells showed higher expression. Functionally, a decrease in adhesion to human umbilical vein endothelial cells was observed in the differentiated Caco-2 cells. Thus, regulation of ICAM-1 and VCAM-1, although both NF-jB target genes, appears to be different over the course of epithelial differentiation in Caco-2 cells. Introduction The intestinal epithelium is formed of a single layer of columnar cells that invaginate as crypts into the underlying connective tissue. These crypts house multipotent stem cell niches, which differentiate into absorptive cells, mucus-producing goblet cells or endocrine cells as they move upwards towards the villi to be finally expelled into the lumen [1]. As the cells differentiate, they lose their ability to proliferate and form tight junctions that act as a physical barrier to bacteria while allowing selective absorption from the gut lumen Abbreviations ALLN, N-[N-(N-Acetyl-L-leucyl)-L-leucyl]-L-norleucine; C EBPb, CCAAT enhancer binding protein b; DN, dominant-negative; EMSA, electrophoretic mobility shift assay; EV, empty vector; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HUVECs, human umbilical vein endothelial cells; ICAM-1, intercellular cell adhesion molecule 1; IjB, inhibitor of jb; LFA1, lymphocyte function-associated antigen 1; NAC, N-acetyl cysteine; NF-jB, nuclear factor jb; NR, non-related; PDTC, pyrrolidine dithiocarbamate; PKC, protein kinase C; TMB-8, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester; VCAM-1, vascular cell adhesion molecule 1; VLA1, very late antigen FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

2 E. Astarci et al. ICAM-1 and VCAM-1 regulation in differentiation [2]. These cells are also located in close proximity to endothelial cells, which makes them important for signal transduction in the immunological response of the gut [3]. Intercellular cell adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) are involved in adhesion of leukocytes as well as metastatic cancer cells to endothelial cells [4,5]. In inflammatory bowel disease, as well as in colon tumors, increased expression of these adhesion molecules is involved in the maintenance of chronic inflammation [6,7]. Additionally, increased expression of ICAM-1 and VCAM-1 in resected human colon carcinoma cells compared to matched normal tissues has been reported [8]. Transcriptionally, both ICAM-1 and VCAM-1 are regulated by nuclear factor jb (NF-jB) [4]. NF-jB is an inflammatory transcription factor that is frequently dysregulated in many cancers, including colorectal cancer, leading to increased cellular transformation, proliferation and loss of apoptosis [9,10]. In the absence of a stimulus, the NF-jB subunits bind to inhibitor of jb (IjB) and are held in the cytoplasm [11]. Following a pro-inflammatory stimulus, IjB is phosphorylated by inhibitor of NF-jB kinase, allowing it to be degraded in the proteasome, thereby freeing the NF-jB subunits (most commonly p65 and p50) for nuclear localization and binding to the promoter regions of target genes [9,12]. However, several studies have indicated differential regulation of ICAM-1 and VCAM-1, indicating the involvement of other signaling pathways [13 16]. One such candidate is CCAAT enhancer binding protein b (C EBPb), which binds to an enhancer element in the promoter of ICAM-1 but not VCAM-1 [17]. C EBPb has been shown to be functional in the differentiation of adipocytes [18], uterine stromal cells [19], mammary epithelial cells [20] and hepatocytes [21]. Additionally, it was also shown that delivery of the full-length C EBPb gene in nude mice suppressed the growth of colonic tumors by inducing apoptosis [22]. The process of intestinal differentiation is accompanied by functional and morphological changes. Differentiated cells in the intestine are characterized by the presence of tight junctions (and thereby reduced permeability) and the development of brush border membranes, together with expression of sucrase isomaltase, alkaline phosphatase and lactase [23,24]. A leaky intestinal epithelium, characterized by increased paracellular permeability, is associated with increased inflammatory NF-jB activity and is a hallmark of inflammatory bowel disease [25]. It has been reported that colon cells undergoing differentiation induced by sodium butyrate (a histone deacetylase inhibitor) show decreased activation of NF-jB [26 28]. In the absence of any commercially available immortalized normal human colon epithelial cell line that can differentiate in vitro, the Caco-2 cancer cell line, which can spontaneously re-differentiate into enterocyte type cells, has been widely used as a model for intestinal differentiation [23]. Given the intriguing observation of a decrease in VCAM-1 mrna expression and an increase in ICAM-1 mrna expression, together with a decline in the protein expression of both ICAM-1 and VCAM-1 over the course of spontaneous differentiation of Caco-2 cells, we wished to examine the regulation of ICAM-1 and VCAM-1 at the transcriptional, post-transcriptional and post-translational levels. We have shown here for the first time that the process of spontaneous differentiation entails differential transcriptional regulation of ICAM-1 and VCAM-1. Posttranslationally, the ICAM-1 protein is degraded in the proteasome and lysosome, and an overall decrease in adhesion to endothelial cells was observed in differentiated Caco-2 cells. Moreover, a decrease in ICAM-1 protein was observed in moderately and poorly differentiated grade II and grade III human colon tumors compared to matched normal well-differentiated tissues. These data provide us with insights into how the process of differentiation may contribute to the regulation of cell adhesion genes. Results Caco-2 cells were originally isolated from a well-differentiated colon tumor and shown to undergo re-differentiation into enterocyte type cells with brush borders spontaneously upon reaching confluency [23]. The differentiated cells characteristically are growth-arrested, and express brush border hydrolases such as alkaline phosphatase. We grew Caco-2 cells over several days after reaching confluency, and monitored the alkaline phosphatase activity qualitatively by an NBT BCIP staining method, and quantitatively by a colorimetric assay using p-nitrophenyl phosphate substrate over the course of differentiation (Fig. 1A). Both assays indicated higher alkaline phosphatase activity in differentiated Caco-2 cells compared to undifferentiated cells. Villin is a Ca 2+ ion-regulated protein that binds to actin in the cytoskeleton of the brush border microvilli and is expressed in differentiated Caco-2 cells [29]. Using quantitative RT-PCR, villin mrna was found to be expressed at 7 10-fold higher levels in Caco-2 cells grown for 7 30 days after reaching confluency compared to undifferentiated cells (Fig. 1B). Moreover, we observed an increase in the protein levels of carcinoembryonic antigen, a well-established FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 2967

3 ICAM-1 and VCAM-1 regulation in differentiation E. Astarci et al. Fig. 1. Confirmation of differentiation and expression of ICAM-1 and VCAM-1 in spontaneously differentiating Caco-2 cells. (A) Alkaline phosphatase staining (left panels) and alkaline phosphatase activity (right panel). (B) Villin expression by quantitative RT-PCR and (C) protein expression of carcinoembryonic antigen (CEA), for confirmation of differentiation in Caco-2 cells. (D) VCAM-1 (left panel) and ICAM-1 (right panel) mrna expression. (E) Western blot showing the protein levels of both ICAM-1 and VCAM-1 during differentiation of Caco-2 cells. GAPDH was used as a loading control. Statistical comparisons were carried out using ANOVA with Tukey s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). marker of differentiation in Caco-2 cells [30], from day 5 onwards after reaching 100% confluency (Fig. 1C). These assays confirm the induction of postconfluence spontaneous differentiation in Caco-2 cells. ICAM-1 and VCAM-1 are differentially expressed over the course of differentiation of Caco-2 cells Post-confluent Caco-2 cells collected at various days of differentiation were assayed for VCAM-1 and ICAM-1 mrna expression by quantitative RT-PCR. We observed that VCAM-1 mrna expression decreased over time, with a significant (P < 0.05) decrease in expression 20 days after reaching confluency (Fig. 1D, left panel). Interestingly, ICAM-1 mrna expression increased over the 14-day culture period after reaching confluency (Fig. 1D, right panel). However, both ICAM-1 and VCAM-1 protein levels decreased over the course of spontaneous differentiation (Fig. 1E). Transcriptional regulation of VCAM-1 and ICAM-1: role of NF-jB Having observed the discrepancy in mrna expression of two NF-jB target genes, i.e. a decrease in VCAM-1 mrna expression and an increase in ICAM-1 mrna expression, we next determined whether the process of cellular differentiation in Caco-2 cells affected NF-jB activity. To achieve this, we performed a number of assays on Caco-2 cells at day 0 (undifferentiated) and day 10 (differentiated) after they reached 100% confluence. NF-jB is normally retained in the cytoplasm by IjBa. For nuclear translocation of NF-jB, IjBa is phosphorylated by inhibitor of NF-jB kinase and degraded in the proteasome. We therefore determined the phosphorylation of cytoplasmic IjBa by western blot and nuclear translocation of RelA p65 by western blot and immunofluorescence in Caco-2 cells. We 2968 FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

4 E. Astarci et al. ICAM-1 and VCAM-1 regulation in differentiation observed decreased phosphorylation of IjBa at Ser32 36 in the differentiated Caco-2 cells compared to the undifferentiated cells (Fig. 2A). However, in contrast to our expectations, we did not observe a dramatic increase in the levels of total IjBa in differentiated cells. An interesting target gene of NF-jB is IjBa itself. It has been shown that active NF-jB increases the expression of IjBa such that its own activity is controlled tightly in a negative feedback manner in non-transformed cells [31] and cancer cells [32]. It was reported by Scott et al. [32] that, when NF-jB is active and there is more p65 in the nucleus, more total IjBa is produced. The reason why there is no obvious change in the total levels of IjBa may be the higher expression of IjBa in undifferentiated cells (day 0) in the presence of active p65. Therefore, in undifferentiated cells, IjBa is phosphorylated and degraded, and NF-jB can freely enter the nucleus and induce expression of IjBa. This is indicated by the strong band of total IjBa observed in the undifferentiated day 0 cells. On the other hand, in the differentiated cells, IjBa is phosphorylated less, and therefore degraded less, so NF-jB is held in the cytoplasm with no new expression of IjBa. Therefore, the total levels of IjBa may be kept more or less constant in the two differentiation states of the cells by different mechanisms. An immunofluorescence assay using Caco-2 cells that had been allowed to differentiate showed a significant (P < 0.01) decrease in the nuclear to cytoplasmic ratio of both p65 and p50 in the differentiated cells compared to the undifferentiated cells, indicating lower nuclear translocation of NF-jB. Moreover, western blotting using nuclear and cytoplasmic extracts from day 0 and day 10 Caco-2 cells also showed decreased nuclear levels of p65, together with a marked decrease in the phosphorylation of total p65 protein at Ser536 after induction of differentiation (Fig. 2B). We next determined whether the loss of nuclear translocation of NF-jB was also associated with loss of the DNA-binding ability of NF-jB. To achieve this, we performed a non-radioactive electrophoretic mobility shift assay (EMSA) using nuclear extracts from undifferentiated and differentiated Caco-2 cells and an oligonucleotide containing the NF-jB consensus sequence (Fig. 2C). We observed a slight decrease in DNA binding in differentiated cells compared with undifferentiated cells (lane 3 versus lane 2). The specificity of the reaction was confirmed by incubating the reaction mixture with a 200-fold excess of the cold probe, resulting in a loss of mobility shift (Fig. 2C, lane 4), as well as by incubation with 2 ll of p65 and p50 antibodies, which resulted in a supershift (Fig. 2C, lanes 5 and 6, respectively). In order to confirm this data, we performed an ELISA assay to determine NF-jB consensus sequence binding by nuclear extracts from day 0 and day 10 Caco-2 cells (Fig. 2D). We again observed a significant decrease in the DNA-binding activity of both p50 (P < ) and p65 (P = ) NF-jB proteins in the differentiated cells (day 10), compared to the undifferentiated cells (day 0). Furthermore, treatment of the differentiated (day 0) cells with SN50, a synthetic 41- amino acid peptide NF-jB inhibitor, resulted in a significant decrease in the DNA-binding activity of both p65 (P = ) and p50 (P < ). In order to confirm the recruitment of NF-jB to the endogenous promoters of ICAM-1 and VCAM-1, chromatin immunoprecipitation (ChIP) was performed using genomic DNA from undifferentiated (day 0) and differentiated (day 10) Caco-2 cells (Fig. 2E). In the presence of the p65 antibody, both ICAM-1 and VCAM-1 promoters were precipitated from formaldehyde cross-linked lysates of undifferentiated cells. A significant decrease in the amplified signal was observed by semi-quantitative and quantitative PCR in day 10 cells, indicating a loss of NF-jB recruitment to both ICAM-1 and VCAM-1 promoters in the differentiated Caco-2 cells. The 5 upstream regions of both ICAM-1 and VCAM-1 promoters, which do not contain any transcription factor binding sequences, were not precipitated by the p65 antibody, and served as controls. We next determined the transcriptional activity of NF-jB over the course of spontaneous differentiation of Fig. 2. NF-jB is inhibited in the course of differentiation. (A) Phosphorylation of IjBa in differentiated (D10) cells compared to undifferentiated (D0) cells. (B) Left upper panel: Nuclear (N) and cytoplasmic (C) p65; left lower panel: phosphorylation of p65 in D10 cells compared to D0 cells. Middle panel: Immunofluorescence for p50 and p65 in Caco-2 cells. Right panel: Quantification of the immunofluorescence data showing the nuclear to cytoplasmic ratio of p50 and p65. Asterisks indicate a statistically significant difference compared with day 0 (P < 0.01, nested ANOVA). (C) EMSA and (D) ELISA for NF-jB DNA binding in D10 cells compared to D0 cells. Asterisks indicate statistically significant differences (t test). (E) Chromatin immunoprecipitation for NF-jB binding to the promoter of VCAM-1 and ICAM-1 in differentiated Caco-2 cells by semi-quantitative and quantitative PCR. Asterisks indicate a statistically significant difference compared with day 0 (t test). (F) Luciferase assay for NF-jB transcriptional activity in D10 cells compared to D0 cells. Asterisks indicate a statistically significant difference (ANOVA with Tukey s post hoc test). (G) Luciferase assay for NF-jB with treatment of day 0 cells with various NF-jB inhibitors compared to untreated cells (UT). (H) Expression of ICAM-1 and VCAM-1 proteins after TMB-8 treatment (left panel) and ELISA-based DNA binding assays for NF-jB (middle and right panels) in day 0 cells treated with TMB-8. FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 2969

5 ICAM-1 and VCAM-1 regulation in differentiation E. Astarci et al FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

6 E. Astarci et al. ICAM-1 and VCAM-1 regulation in differentiation Caco-2 cells. To achieve this, the NF-jB consensus sequence cloned into the pgl3 vector was transfected into Caco-2 cells at day 0 and day 10 after reaching confluency. Controls included a non-related (NR) counterpart (in which most of the transcription factor consensus sequence was changed to cytosines) and the empty pgl3 vector (see Table S1 for sequences). We observed a significant (P = ) decrease in the luciferase activity when the NF-jB luciferase vector, but not the NR or the empty pgl3 (EV) vectors, was transfected into differentiated cells compared to undifferentiated cells (Fig. 2F). This indicates a loss of NF-jB transcriptional activity in the differentiated Caco-2 cells. In order to further validate the luciferase assay results, undifferentiated (day 0) Caco-2 cells were incubated with several known NF-jB inhibitors, including the antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetyl cysteine (NAC), the synthetic peptide SN50 and the intracellular Ca 2+ chelator TMB-8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester] [33,34]. As expected, we observed a significant decrease in luciferase activity in the presence of all inhibitors (Fig. 2G). A loss of NF-jB activity was observed in the presence of TMB-8, which prevents release of Ca 2+ from intracellular stores [35], indicating a potential role for Ca 2+ -dependent pathways in regulation of NF-jB. Further confirmation of the inhibitory role of TMB-8 was established by loss of NF-jB DNAbinding activity and a decrease in expression of both NF-jB target proteins VCAM-1 and ICAM-1 in undifferentiated confluent (day 0) Caco-2 cells (Fig. 2H). NF-jB transactivation by PKCa and PKCh is inhibited in differentiated Caco-2 cells As treatment of the cells with the Ca 2+ antagonist TMB-8 inhibited NF-jB, we hypothesized that a conventional Ca 2+ -dependent protein kinase C (PKC) may be involved in NF-jB activation. We first performed a non-radioactive PKC kinase assay in which proteins were extracted from spontaneously differentiating Caco-2 cells, incubated with a fluorescent peptide specific for PKC, and electrophoresed on an agarose gel. Phosphorylation of the peptide (due to the kinase activity of PKC) results in a change in the charge, allowing separation on an agarose gel at neutral ph (Fig. 3A). For quantitative analysis, the phosphorylated peptide bands observed in the gel were cut, solubilized and measured spectrophotometrically. We observed a significantly lower phosphorylation of the peptide in the day 10 differentiated cells (P = ) compared to the day 0 undifferentiated cells, indicating that PKC was less active in the differentiated cells. Additionally, in order to determine the specific subtype of PKC that was involved in this activation axis, a PKCa dominantnegative (PKCa-DN) vector was transfected into the undifferentiated day 0 Caco-2 cells. Inhibition of PKCa resulted in a significant (P = ) decrease in phosphorylation of the peptide. Further proof of activation of PKCa was obtained by transfection of the day 10 differentiated cells with a PKCa over-expressing vector. Recovery of phosphorylation of the peptide was observed in the differentiated cells when PKCa was over-expressed, thereby confirming that the kinase activity of PKCa decreases over the course of spontaneous differentiation of Caco-2 cells. PKCa has previously been shown to activate NF-jB via PKCh, a novel Ca 2+ -independent member of the PKC family of proteins that is active at the immunological synapse formed by T cells [36]. PKCh is also expressed in Caco-2 cells [37]. We therefore hypothesized that PKCa may activate PKCh, which in turn may be involved in activation of NF-jB in Caco-2 cells. Phosphorylation of PKCh at Thr538 was shown to be necessary for the activation of the inhibitor of NF-jB kinase in Jurkat T cells [38]. Therefore, we probed Caco-2 whole-cell extracts from undifferentiated confluent (day 0) and differentiated (day 10) cells using an antibody that recognizes PKCh phosphorylated at Thr538. We observed a decrease in the phosphorylation of PKCh, implying a decrease in its activity, in the differentiated cells compared to the undifferentiated cells (Fig. 3B). In order to establish activation of PKCh by PKCa, we transfected undifferentiated (day 0) Caco-2 cells separately with the PKCa over-expressing vector and the PKCa-DN vector. Increased phosphorylation of PKCh was observed in the PKCa over-expressing cells, and a loss of PKCh phosphorylation was observed in the cells transfected with the PKCa-DN vector (Fig. 3C). Next, we wanted to establish whether any cross-talk exists between PKCa and NF-jB. First, we determined the phosphorylation of IjBa in the presence of PKCa over-expression or inhibition by the PKCa-DN vector (Fig. 3D). We observed an increase in IjBa phosphorylation when PKCa was over-expressed, indicating activation of NF-jB. A loss of IjBa phosphorylation in the cells transfected with the PKCa-DN vector indicated that NF-jB was retained in the cytoplasm in these cells and thus rendered inactive. We then determined whether PKCa and PKCh affected the DNA-binding ability of NF-jB. To achieve this, we transfected the undifferentiated (day 0) cells with the PKCa-DN vector or treated the cells with the PKCa b inhibitor Go 6976 [39] or the PKCh inhibitor rottlerin [40] (Fig. 3E). An ELISA-based FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 2971

7 ICAM-1 and VCAM-1 regulation in differentiation E. Astarci et al. Fig. 3. PKCa activates NF-jB via PKCh in Caco-2 cells. (A) PKC kinase activity in D10 cells compared to D0 cells. DN; dominant-negative, Oex; over-expression. (B) Phosphorylation of PKCh at Thr538 in D10 cells compared to D0 cells. (C,D) Levels of phosphorylated PKCh (C) and phosphorylated IjBa (D) in PKCa over-expressing and PKCa-DN-expressing D0 Caco-2 cells. (E) NF-jB DNA binding in D0 Caco-2 cells expressing PKCa-DN vector or treated with the PKCa b inhibitor Gö6976 or the PKCh inhibitor rottlerin. Statistical comparisons are with respect to the Day 0 bar. (F) Luciferase assay for NF-jB in Caco-2 cells over-expressing PKCa or transfected with PKCa-DN vector compared to cells transfected with the NF-jB luciferase vector alone. NR, non-related consensus sequence; EV, empty pgl3 vector. Statistical significance was determined by one-way ANOVA using Tukey s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). NF-jB DNA-binding assay indicated a significant reduction in NF-jB DNA binding when either PKCa or PKCh were inhibited. To ascertain whether the PKCa PKCh axis also affected the transcriptional activity of NF-jB, reporter gene assays were performed. Caco-2 cells were grown to confluency, and, on day 0 or day 10, were transfected with either the NF-jB pgl3 luciferase vector, the non-related (NR) counterpart, or the empty pgl3 vector. At the same time, the cells were co-transfected with the PKCa over-expression vector or the PKCa- DN vector (Fig. 3F). The transcriptional activity of NF-jB in the undifferentiated (day 0) Caco-2 cells was significantly (P < ) higher when PKCa was over-expressed and significantly (P < ) lower when the cells were co-transfected with the PKCa-DN vector compared to cells transfected with the NF-jB luciferase vector alone. As expected, the NF-jB transcriptional activity was always lower in the differentiated cells (day 10) than the undifferentiated cells (day 0) irrespective of over-expression or inhibition of PKCa. A similar decrease in the NF-jB transcriptional activity of the undifferentiated cells (day 0) was observed when the cells were treated with the PKCa and PKCh inhibitors Go 6976 and rottlerin (data not shown). Taken together, we have shown here that one of the pathways of NF-jB activation in undifferentiated Caco-2 cells is through activation of PKCa and PKCh, and that this pathway is inhibited in differentiated cells. ICAM-1 mrna expression is transcriptionally regulated by C/EBPb in differentiated cells The initial expression analysis of the NF-jB target genes VCAM-1 and ICAM-1 indicated that, unlike the VCAM-1 mrna, ICAM-1 mrna expression increases over the course of differentiation (Fig. 1D). Having 2972 FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

8 E. Astarci et al. ICAM-1 and VCAM-1 regulation in differentiation established that NF-jB activation and transcriptional activity were decreased in the differentiated cells compared to undifferentiated cells (see above), we then sought an explanation for the increased ICAM-1 mrna expression over the course of differentiation. ICAM-1, but not VCAM-1, is reported to be closely regulated by the transcription factor C EBPb [17,41,42]. We first established whether the nuclear levels of C EBPb changed over the course of differentiation. Nuclear extracts from undifferentiated (day 0) and differentiated (day 10) cells probed with a C EBPb antibody indicated an increase in nuclear C EBPb in the differentiated cells (Fig. 4A). In order to determine whether the DNA-binding activity of C EBPb increased over the course of differentiation, we performed a non-radioactive EMSA using nuclear extracts from undifferentiated and differentiated Caco-2 cells and an oligonucleotide containing the C EBPb consensus sequence. We observed (Fig. 4B) a dramatic increase in DNA-bound C EBPb in day 10 differentiated cells (lane 3) compared to undifferentiated cells (lane 2). The specificity of the reaction was ensured by incubating the reaction mixture with a 200-fold excess of the cold probe, which resulted in a loss of mobility shift (lane 4). as well as by incubation with 2 ll of the C EBPb antibody, which resulted in a supershift (lane 5). In order to further confirm recruitment of C EBPb to the endogenous promoters of ICAM-1, ChIP was performed using genomic DNA from undifferentiated (day 0) and differentiated (day 10) Caco-2 cells. The ICAM-1 promoter has two C EBPb binding sites. One is a composite C EBPb NF-jB binding site at position )178 relative to the transcription start site, which contains consensus sequences for both C EBPb and NF-jB binding, and binding of both transcription factors has been shown to be necessary for ICAM-1 transcription in HeLa cells [41]. In addition, there is a second C EBPb binding site at position )364. C EBPb recruitment to both sites was analyzed by ChIP (Fig. 4C). Quantitative RT-PCR indicated a significant increase (P < ) in precipitation of C EBPb from the chromatin of differentiated compared to the undifferentiated Caco-2 cells, indicating that the transcription factor is recruited to the promoter of ICAM-1 to a greater extent when the cells are differentiated. Moreover, we observed that, in differentiating Caco-2 cells, C EBPb is recruited to the single C EBPb consensus site, rather than the C EBPb NF-jB composite element. Therefore, unlike HeLa cells [41], Fig. 4. C EBPb transcriptional activity increases in differentiated Caco-2 cells and transcriptionally up-regulates ICAM-1. (A) C EBPb protein in nuclear (N) and cytoplasmic (C) extracts of differentiated Caco-2 cells (D10) compared with undifferentiated cells (D0). (B) EMSA for C EBPb in D10 cells compared to D0 cells. (C) Chromatin immunoprecipitation for recruitment of C EBPb to the promoter of ICAM-1 in Caco-2 cells by semi-quantitative and quantitative PCR (t test). (D) Luciferase assay for C EBPb transcriptional activity in D10 differentiated cells compared to D0 cells (ANOVA). (E) ICAM-1 protein levels in D10 Caco-2 cells transfected with a C EBPb dominant-negative construct (D10 DN) compared to untransfected D10 or D0 cells. FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 2973

9 ICAM-1 and VCAM-1 regulation in differentiation E. Astarci et al. differentiating Caco-2 cells do not require cooperative recruitment of both C EBPb and NF-jB to induce transcription of ICAM-1. This further shows that the two transcription factors may function independently; in this case, with the activity of NF-jB decreasing and that of C EBPb increasing over the course of differentiation of Caco-2 cells. To determine whether the transcriptional activity of C EBPb was altered over the course of differentiation of Caco-2 cells, we cloned the C EBPb consensus sequence from the promoter of ICAM-1, upstream of a luciferase reporter gene in the pgl3 vector (see Table S1 for sequences). The pgl3 vector cloned with a non-related (NR) sequence, in which the C EBPb consensus sequence was changed to cytosines, and the empty vector (EV) served as controls. We transfected these vectors separately into undifferentiated (day 0) and differentiated (day 10) Caco-2 cells. We observed (Fig. 4D) a significantly (P < 0.05) higher transcriptional activity of C EBPb in the differentiated cells (day 10) than in undifferentiated confluent cells (day 0). As expected, no difference in luciferase activity was observed when cells at day 0 or day 10 were transfected with the NR or EV controls. In order to confirm the involvement of C EBPb in expression of ICAM-1 in the differentiated cells, we transfected the day 10 differentiated cells with a dominant-negative construct of C EBPb (C EBPb-DN). This plasmid expresses a C-terminal 20 kda form of C EBPb, which has a dominant-negative function against the larger active forms of C EBPb [42]. Expression analysis of ICAM-1 mrna from day 10 Caco-2 cells transfected with this construct indicates that C EBPb-DN successfully inhibited expression of ICAM-1 mrna, whereas day 10 cells not transfected with the construct showed robust expression of ICAM-1 mrna (Fig. 4E). Taken together, our data indicate that, in the differentiated Caco-2 cells, it is likely that transcription of ICAM-1 is largely regulated by the activity of C EBPb. Post-transcriptional regulation of ICAM-1 Fig. 5. ICAM-1 is not regulated by micrornas in the course of spontaneous differentiation of Caco-2 cells. The 3 UTR of the ICAM-1 gene was cloned in two overlapping parts (1.1 and 1.2) into a pmir REPORT vector. Based on the observation of increased ICAM-1 mrna expression, together with a decrease in the protein expression of ICAM-1, we hypothesized that micrornas (mirnas) may be involved in post-transcriptional regulation of ICAM-1 expression. mirnas are short bp non-coding RNAs that can regulate gene expression, usually by translational repression [43]. To assess their involvement, we cloned the entire 3 UTR of the ICAM-1 gene in two halves into a pmir REPORT luciferase vector, with a 150 bp overlap between the two constructs. The vectors were then transfected into undifferentiated (day 0) or differentiated (day 10) Caco-2 cells (Fig. 5). The existence of mirna regulation is indicated by a reduction in the luciferase activity. However, no such reduction was observed in the day 0 or day 10 cells, indicating that ICAM-1 is most likely not regulated by mirnas in differentiating Caco-2 cells. Post-translational regulation of ICAM-1 In order to explain the loss of protein expression in ICAM-1 despite robust mrna expression and no apparent mirna-mediated regulation, we hypothesized that a protein degradation pathway may have been activated. Using specific inhibitors, we analyzed three major pathways through which proteins are degraded in cells: the 26S proteasomal, lysosomal and calpain 1-mediated pathways [44]. We treated undifferentiated (day 0) and differentiated (day 10) confluent cells with inhibitors for the respective pathways for 24 h, before protein extraction and western blotting using an ICAM-1 antibody (Fig. 6). Treatment of the cells with the proteasomal inhibitor resulted in recovery of the levels of ICAM-1 protein in the differentiated Caco-2 cells. We did not observe a significant recovery of the levels of ICAM-1 protein in the undifferentiated cells, indicating that ICAM-1 protein degradation in the proteasome is most likely specific to the process of differentiation in these cells. ICAM-1 has been previously shown to be degraded in the lysosome [44]. We observed recovery of ICAM-1 protein levels in both differentiated and undifferentiated Caco-2 cells when treated with lysosomal inhibitors, indicating that this degradation pathway is 2974 FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

10 E. Astarci et al. ICAM-1 and VCAM-1 regulation in differentiation No ICAM-1 expression was seen in non-malignant epithelial cells that were well-differentiated. Of the malignant epithelial cells classified as moderately differentiated, 27% had an ICAM-1 score of 0, 55% had a score of 1 and 18% had a score of 2. Of the poorly differentiated malignant epithelial cells, 11% had an ICAM-1 score of 1, 78% had a score of 2, and 11% had a score of 3 (see Experimental procedures). A statistically significant increase in ICAM-1 protein levels was seen in the grade II moderately differentiated tissue (P < compared to well-differentiated tissue, v 2 test) as well as the grade III poorly differentiated tissue (P < compared to well-differentiated tissue, P < compared to moderately differentiated tissue, v 2 test). Functional significance of regulation of cell adhesion molecules in Caco-2 differentiation Fig. 6. ICAM-1 is degraded in the proteasome and lysosome, but not by the calpain-mediated protein degradation pathway in Caco-2 cells. D0 or D10 Caco-2 cells were treated with proteasomal (upper panel), lysosomal (middle panel) or calpain (lower panel) inhibitors, and probed with ICAM-1 antibody. independent of the differentiation status of the cells. However, treatment of Caco-2 cells with the calpain 1 inhibitor did not result in recovery of the levels of ICAM-1 protein in either differentiated or undifferentiated cells, indicating that this pathway of protein degradation is not active in these cells. Immunohistochemistry analysis of ICAM-1 expression in resected colon tumor samples In order to determine whether ICAM-1 protein levels were altered in resected human colorectal adenocarcinoma at various stages of tumor differentiation, tumor tissue from 20 patients was randomly selected from the archives of the Pathology Department of the Ankara University School of Medicine. Hematoxylin & eosin-stained slides were used to grade tumors as recommended by World Health Organization classification of colorectal tumors on the basis of the percentage of gland formation [45]. The matched normal colon tissue was considered as welldifferentiated control tissue. The tumors were then analyzed for ICAM-1 expression by immunohistochemistry in adjacent sections from the same block (Fig. 7 and Table 1). ICAM-1 and VCAM-1 are cell adhesion molecules that are known to be involved in adhesion of cells to vascular endothelial cells [46]. In addition, expression of these proteins is also associated with more invasive cancers [4], and cancer cells expressing ICAM-1 and VCAM-1 can extravasate and mediate metastasis by adhesion to endothelial cells [47]. In order to determine whether the process of differentiation affected the cell cell adhesion mediated by cell adhesion molecules, the adhesion of day 0 (undifferentiated) or day 10 (differentiated) Caco-2 cells to human umbilical vein endothelial cells (HUVECs) was assayed in a co-culture model in which the Caco-2 cells were labeled with a fluorescent dye. The number of adhered Caco-2 cells was measured fluorometrically. In order to confirm that the adhesion of Caco-2 cells to HUVECs was mediated by ICAM-1, undifferentiated (day 0) Caco-2 cells were pre-incubated with either increasing amounts of an ICAM-1 antibody or a non-specific isotypic IgG. As a control, Caco-2 cells were directly added to wells containing ICAM-1 antibody but without pre-coating with the HUVEC monolayer (Fig. 8). The data obtained indicate that adhesion of Caco-2 cells to HUVECs was significantly lower in the differentiated cells compared to the undifferentiated cells (P < 0.001). In the presence of increasing amounts of ICAM-1 antibody (Day 0 + ICAM1 and Day 0 + ICAM1_10), which bound to the ICAM-1 protein and thereby prevented interactions with its ligands on endothelial cells, the undifferentiated Caco-2 cells adhered significantly less (P < 0.001) to the HUVECs. Furthermore, the specificity of the reaction was confirmed by addition of an IgG antibody, which did not lead to any decrease in adhesion. Additionally, both Caco-2 cells FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 2975

11 ICAM-1 and VCAM-1 regulation in differentiation E. Astarci et al. Fig. 7. Immunohistochemistry showing ICAM-1 expression in resected human colon cancer samples and matched normal colon epithelium as a function of tumor differentiation. (A) Well-differentiated normal colon section. (B,C) Moderately differentiated colon sections. (D,E) Poorly differentiated tumors. (F H) Hematoxylin & eosin staining of normal (well-differentiated), moderately differentiated and poorly differentiated colon tumor sections. Table 1. Clinicopathological parameters and ICAM-1 expression in colon cancer samples. ICAM-1 staining Samples n Score of 0 Score of 1 Score of 2 Score of 3 Normal (well differentiated) (100%) Grade II (moderately differentiated) 11 3 (27%) 6 (55%) 2 (18%) 0 (0%) Grage III (poorly differentiated) 9 0 (0%) 1 (11%) 7 (78%) 1 (11%) and HUVECs were necessary for the interaction, as incubation of Caco-2 cells with ICAM-1 antibody in wells that are not pre-coated with HUVECs did not lead to any decrease in cell cell interaction. Discussion The intestinal mucosa undergoes continual renewal, with movement of cells along the crypt villus axis. Cell cell adhesion plays a key role in this process, without compromising the barrier function [48]. Differentiation in colonic epithelial cells is associated with an overall decrease in inflammatory events. Decreased activation of the inflammatory transcription factor NF-jB has been reported in colon cancer cell lines induced to differentiate by short-chain fatty acids [27]. Solanas et al. [49] reported that, in confluent SW480 epithelial cells with well-established adherens junctions, the RelA p65 and b-catenin proteins are inhibited through association with E-cadherin at the membrane. Depletion of E-cadherin, as occurs during epithelial to mesenchymal transition, results in translocation of p65 and b-catenin to the nucleus and the transcription of mesenchymal genes [49] FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

12 E. Astarci et al. ICAM-1 and VCAM-1 regulation in differentiation Fig. 8. Differentiated Caco-2 cells show less adhesion to endothelial cells in a co-culture model with HUVECs (P < , ANOVA). In the present study, we observed an increase in ICAM-1 mrna expression and a decrease in VCAM-1 mrna expression, together with a decrease in the protein levels of both ICAM-1 and VCAM-1 during the process of spontaneous differentiation of Caco-2 cells. ICAM-1 and VCAM-1 are members of the immunoglobulin superfamily of proteins that induce cell cell adhesion by interacting with their ligands LFA1 (Lymphocyte function-associated antigen 1) and VLA1 (Very late antigen-1) on endothelial cells or peripheral blood cells [50]. Through such interactions, ICAM-1 and VCAM-1 are involved not only in leucocyte extravasation in an inflammatory response, but are also reportedly involved in adhesion of cancer cells to the microvasculature and their subsequent metastasis to distant organs, and enhancement of the inflammatory environment at the tumor site [46]. As both genes are established NF-jB target genes [4] and yet appeared to be expressed differentially, we examined the transcriptional, post-transcriptional and post-translational regulation of these genes over the course of differentiation of Caco-2 cells. At the transcriptional regulation level, we examined the activation of NF-jB over the course of spontaneous differentiation of Caco-2 cells. We observed that differentiation was associated with an inhibition of NF-jB nuclear translocation, DNA binding and recruitment to endogenous ICAM-1 and VCAM-1 promoters, as well as transcriptional activity. Vallee et al. [51] reported activation of NF-jB when differentiated Caco-2 cells were treated with interleukin 1b. However, in that study, the Caco-2 cells were differentiated for just 3 days. Our study involves differentiation over at least 10 days after reaching 100% confluency, when all the markers of differentiation are well established. Moreover, we did not use cytokine stimulation, which may account for the differences in the results. We next wished to explore the mechanistic underpinnings of inhibition of NF-jB in the differentiated cells. Treatment of undifferentiated day 0 Caco-2 cells (when NF-jB is active) with TMB-8, an intracellular Ca 2+ chelator, resulted in a significant loss of NF-jB transcriptional activity. A similar loss of activity was also observed with other well-established NF-jB inhibitors. TMB-8 treatment was also accompanied by a loss of VCAM-1 and ICAM-1 protein expression. High dietary intake of Ca 2+ has been shown to promote cellular differentiation in the colon and reduce the risk of colorectal cancer [52]. Moreover, activation of an extracellular Ca 2+ sensor receptor in colon cancer cell lines was reported to increase the expression of E-cadherin and inhibit the expression of b-catenin, together with increasing the expression of differentiation markers [53]. Intracellular Ca 2+ is generally accepted as an important second messenger that can cross-talk with a number of signaling cascades. The various transcription factors that Ca 2+ can activate include calcineurin and its target nuclear factor of activated T cells, and Ca-calmodulin-dependent kinase and its target cyclic AMP-responsive element-binding protein, as well as NF-jB [54,55]. As conventional Ca 2+ dependent PKC has been shown to activate NF-jB activity [56], and calcium chelation by TMB-8 treatment inhibited NF-jB, we hypothesized that PKC is involved in the activation of NF-jB. PKCa has previously been shown to be activated in Caco-2 cells treated with an outer membrane preparation of enteropathogenic Escherichia coli; however, whether NF-jB was activated in these cells or not was not determined [57]. Moreover, PKCh has been shown to act downstream of PKCa in the activation of NF-jB in T cells [40,58]. We have established here for the first time that the kinase activity of both PKCa and PKCh was decreased in the differentiated Caco-2 cells, and that PKCa acted upstream of PKCh. Over-expression of PKCa resulted in activation of NF-jB in both differentiated and undifferentiated Caco-2 cells. On the other hand, inhibition of PKCa by a dominant-negative construct (PKCa-DN) or treatment of undifferentiated Caco-2 cells with specific inhibitors of PKCa and PKCh resulted in loss of NF-jB activation. We have therefore established that, in the undifferentiated cells, NF-jB is activated via a PKCa PKCh signaling axis. This axis was inhibited in the differentiated cells, FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 2977

13 ICAM-1 and VCAM-1 regulation in differentiation E. Astarci et al. resulting in inhibition of NF-jB and thereby expression of VCAM-1 mrna and protein. In order to explain the increase in ICAM-1 mrna expression over the course of differentiation, we searched for a transcription factor that regulates ICAM-1 but not VCAM-1. Vallee et al. [51] reported that levels of endogenous ICAM-1 mrna in polarized (differentiated) Caco-2 cells are independent of the NF-jB pathway, and hypothesized that other transcription factors may be involved [51]. A number of reports in the literature have shown that ICAM-1and VCAM-1 may be regulated differentially [16,59], with emphasis on the contribution of the transcription factor C EBPb to expression of ICAM-1, but not VCAM-1 [17,42,60]. Interestingly, this transcription factor is also involved in the differentiation of a number of cell types [18 21]. We therefore hypothesized that increased activity of C EBPb may contribute towards the increasing mrna levels of ICAM-1 observed over the course of Caco-2 differentiation. We observed increased nuclear levels of C EBPb protein, together with increased DNA binding in the differentiated Caco-2 cells compared to the undifferentiated confluent cells. Recruitment of C EBPb to the endogenous promoter of ICAM-1 was also higher in the differentiated cells. Of interest here, previous reports have suggested that C EBPb and NF-jB bind to a composite element on the ICAM-1 promoter and function cooperatively in regulating expression of the gene [41,61]. However, these transcription factors appear to be disconnected during differentiation in the Caco-2 cells, with NF-jB activity seen to decrease and C EBPb activity seen to increase. When we examined recruitment of transcription factors to the composite C EBPb NF-jB binding element by ChIP, we did not observe any immunoprecipitation of either transcription factor, indicating that these two transcription factors do not work in concert in the course of differentiation of Caco-2 cells. Instead, increased C EBPb recruitment was observed at position )364 from the transcription start site in the promoter of ICAM-1 in the differentiated cells. Additionally, an increase in the C EBPb transcriptional activity was also observed with a luciferase reporter system in the differentiated cells. More importantly, when we transfected the day 10 differentiated cells with a dominantnegative construct of C EBPb [42], we observed lower ICAM-1 mrna expression. Having shown that ICAM-1 up-regulation at the mrna level in the differentiating cells could be attributed to the transcriptional activity C EBPb, we then sought to establish the reason for the loss of ICAM-1 protein over the course of differentiation. mirnas are known to regulate the post-transcriptional expression of genes through translational repression [43]. We therefore cloned the 3 UTR of the ICAM-1 gene into a pmir REPORT vector, which, when transfected into cells, shows a decrease in luciferase activity when there is mirna-mediated regulation. However, we did not observe any change in luciferase activity, indicating that the loss of ICAM-1 protein was most likely not due to mirna regulation. In order to determine whether protein degradation contributed to the loss of ICAM-1 protein in the differentiated cells, we examined the degradation of ICAM-1 via the proteasomal, lysosomal or calpain-mediated pathways using specific inhibitors. We observed recovery of the levels of ICAM-1 protein in differentiated cells only using the proteasomal inhibitor MG132. Although MG132 is also known to inhibit the NF-jB pathway [33], we did not observe any decrease in protein levels of ICAM-1 in response to MG132 in the day 0 undifferentiated cells (when NF-jB is active), indicating that the NF-jB pathway was not inhibited at the concentration used. On the other hand, treatment of Caco-2 cells with lysosomal inhibitors resulted in recovery of the levels of ICAM-1 in both differentiated and undifferentiated cells, indicating that this is a constitutively active pathway for the degradation of ICAM-1 in Caco-2 cells. The calpain 1-mediated pathway appeared not to be involved in this degradation process. ICAM-1 protein levels have previously been shown to increase in Caco-2 cells differentiated for a period of 3 days and in the presence of inflammatory cytokines [51]. In the present study, we did not use any cytokines; and moreover examined the molecular changes in Caco-2 cells differentiated over a period of 10 days after achieving confluency. Additionally, use of the relatively high concentrations of cytokines may not be physiologically relevant, which is why determining the ICAM-1 expression in resected human colon samples at various stages of differentiation is critical [62]. Immunohistochemistry analysis of 20 human colorectal adenoncarcinoma resection samples assigned as moderately differentiated and poorly differentiated, showed a distinct increase in ICAM-1 expression as the cells became progressively de-differentiated. Matched normal colon sections, assigned as well-differentiated, did not show any ICAM-1 expression. Although there is a general consensus in the literature regarding lack of expression of ICAM-1 in noninflamed normal colon tissue [51,62,63], a number of reports indicate that ICAM-1 expression and its correlation with tumor metastasis is far from resolved. While ICAM-1 in expression in human malignant cells was generally reported to be associated with metasta FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS