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ß-Catenin triggers nuclear factor ?B-dependent up-regulation of hepatocyte inducible nitric oxide synthase
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The International Journal of Biochemistry & Cell Biology 40 (2008) 1861–1871
-Catenin triggers nuclear factor B-dependent up-regulation of hepatocyte inducible nitric oxide synthase Andrea Bandino, Alessandra Compagnone, Vittoria Bravoco, Carlo Cravanzola, Anna Lomartire, Chiara Rossetto, Erica Novo, Stefania Cannito, Lorenzo Valfr`e di Bonzo, Elena Zamara, Riccardo Autelli, Maurizio Parola, Sebastiano Colombatto ∗ Dipartimento di Medicina e Oncologia Sperimentale, University of Torino, Torino, Italy Received 21 November 2007; received in revised form 18 January 2008; accepted 21 January 2008 Available online 7 February 2008
Abstract Disruption of cell-to-cell contacts, as observed in many pathophysiological conditions, prime hepatocytes for compensatory hyperplastic response that involves induction of several genes, including proto-oncogenes and other gene targets of -catenin signaling pathway. By using cultured hepatocytes and experimental models of adherens junction disruption we have investigated changes in -catenin subcellular localization and their relationships with inducible nitric oxide synthase (iNOS) expression. Two experimental models were employed: (a) rat hepatocytes obtained by collagenase liver perfusion within the first 48 h of culture; (b) 48-h old cultured hepatocytes, transiently transfected or not with a plasmid encoding for dominant/negative inhibitory kappa B␣, exposed to ethylene glycol-bis-(2-aminoethylether)-N,N,N ,N -tetraacetic acid/LiCl treatment. -Catenin signaling and cellular localization, iNOS expression and nuclear factor B involvement, were investigated using morphological, cell and molecular biology techniques. E-cadherin-mediated disruption of cell-to-cell contacts induces early -catenin translocation from membrane to cytoplasm and nuclear compartments, events that are followed by up-regulation of c-myc, cyclin D1 and -transducin repeatcontaining protein expression. This, in turn, resulted eventually in iNOS induction that was mechanistically related to nuclear factor B activation, as unequivocally shown in cells expressing dominant negative inhibitory kappa B-␣. Our data indicate that E-cadherin disassembly and concomitant inactivation of glycogen synthase kinase-3 result in nuclear factor B-dependent induction of iNOS in hepatocytes. © 2008 Published by Elsevier Ltd. Keywords: Lithium; -TrCp1; IB-␣; -Catenin
1. Introduction -Catenin is a multifunctional protein involved mainly in cadherin-mediated cell–cell adhesion (Barth, ∗
Corresponding author at: Dipartimento di Medicina e Oncologia Sperimentale, Sezione di Biochimica, Via Michelangelo 27, 10126 Torino, Italy. Tel.: +39 011 670 5308; fax: +39 011 670 5311. E-mail address: [email protected] (S. Colombatto). 1357-2725/$ – see front matter © 2008 Published by Elsevier Ltd. doi:10.1016/j.biocel.2008.01.029
Nathke, & Nelson, 1997) and in the control of growth and differentiation of several tissues during foetal and adult life. -Catenin, once detached from cytoplasmic domain of E-cadherins, can act as intracellular signal intermediate of the Wnt/wingless pathway (Cadigan & Nusse, 1997; Polakis, 2000) in central nervous system, epidermis (Huelsken, Vogel, Erdmann, Cotsarelis, & Birchmeier, 2001), intestine (Peifer, 2002) and liver (Monga et al., 2003).
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In unstimulated cells, cytoplasmic levels of free -catenin are negatively controlled by means of ubiquitination and subsequent degradation by the 26S proteasome. Glycogen synthase kinase-3 (GSK-3), which is active when complexed with axin, casein kinase 1 (CK1) and the adenomatous polyposis coli gene product (APC), plays a crucial role in ubiquitination by phosphorylating -catenin on 41-threonine residue and multiple serine residues (Ser 33, 37, 61). Activation of the Wnt signal transduction pathway results from the binding of one of the several known components of Wnt-family of extracellular proteins to the receptors Frizzled/LPR5/6. This promotes the disruption of the complex GSK-3–axin–CK1–APC, prevents -catenin phosphorylation and degradation, resulting in increased cytoplasmic levels of -catenin that, in turn, translocates into the nucleus to bind factors TCF/LEF and promote their activation. The latter complex induces transcription of several target genes, including c-myc (He et al., 1998), cyclin D1 (Shtutman et al., 1999), c-jun, fra-1, uPAR (Mann et al., 1999), CD44 (Wielenga et al., 1999), VEGF (Zhang, Phiel, Spece, Gurvich, & Klein, 2003), fibronectin (Gradl, K¨uhl, & Wedlich, 1999), cyclooxygenase-2 (Haertel-Wiesmann, Liang, Fantl, & Williams, 2000) and -transducin repeat-containing protein (-TrCp1) (Spiegelman et al., 2000). -TrCp1, a F-box protein component of a SKp1/Cul1/F-box-type ubiquitin ligase complex able to bind phosphorylated and unphosphorylated target molecules, selectively recognizes only phosphorylated forms of the nuclear factor kappa B (NF-B) inhibitor (IB-␣) and -catenin (Nakayama et al., 2003). -TrCp1 gene expression is then controlled by -catenin (Spiegelman et al., 2000) and it represents a direct link between -catenin and NF-B activation. Primary cultures of rat hepatocytes are a reliable experimental model widely used for studies of tissue engineering, metabolic pathways and signal transduction. Isolation procedure requires ethylene glycol-bis-(2-aminoethylether)-N,N,N ,N -tetraacetic acid (EGTA)/collagenase-dependent disgregation of liver parenchyma and also disrupts cell–cell interactions, rapidly recovered by hepatocytes within 24/36 h of primary culture. This procedure can effectively trigger G0/G1 transition and induce cell cycle-dependent immediate–early proto-oncogenes, then mimicking what is likely to occur during a number of pathophysiological conditions, including partial hepatectomy, necrotic injury, metabolic stress and, more generally, conditions resulting in disruption of cell-to-cell contacts or alterations of extracellular matrix (Etienne et al., 1988; Ikeda, Sawada, Fujinaga, Minase, & Mori, 1989;
Liu, Mars, Zarnegar, & Michalopoulos, 1994; Sawada, 1989). Freshly isolated – and early cultured – hepatocytes have been reported, even in the absence of mitogens, to undergo up-regulation of immediate/early oncogenes (Loyer et al., 1996), most of them being targets of the -catenin/TCF/LEF complex, as well as of inducible nitric oxide synthase (iNOS). iNOS induction, which is a common finding in pathophysiological conditions, is prominent and transient during the first 24 h of culture and leads to accumulation of nitrites in culture medium (Pittner & Spitzer, 1993; Vargiu, Belliardo, Cravanzola, Grillo, & Colombatto, 2000; Vernia, Beaune, Coloma, & Lopez-Garcia, 2001). Moreover, iNOS may be upregulated by isolation-related stimuli like oxidative stress, endotoxins (LPS or lipotheicoic acid) in collagenase preparations and/or release of proinflammatory cytokines (Paine & Andreakos, 2004). In the present study we report that both early cultured hepatocytes as well as stabilized cells treated with the Wnt-mimicking stimulus EGTA/LiCl, used as models of disruption of cell-to-cell contacts, undergo a sequence of events that mechanistically links -catenin nuclear translocation to iNOS induction through NF-B activation. 2. Materials and methods 2.1. Materials Peroxidase- and Cy3-coniugated secondary antibodies were from Amersham (Little Chalfont, Buckinghamshire, UK), horse serum from SPA (Milan, Italy), collagenase, penicillin/streptomycin, M199, insulin, dexamethasone, indirubin-3 -oxime, LiCl, phenylmethylsulfonyl fluoride, Hoechst 33258 and all the other chemicals from Sigma Chemical Co. (St. Louis, MO, USA). All antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) except anti-NOS II (Transduction Laboratories, Lexington, KY, USA) and anti-phospho-GSK-3 (Cell Signaling Technology Inc., Danvers, MA, USA). 2.2. Experimental animals Hepatocytes were isolated by means of collagenase/ perfusion method from male Wistar rats (150–200 g) that were used according to national and local guidelines. Hepatocytes were plated (7 × 104 cells/cm2 ) on culture dishes coated with rat tail tendon collagen (Vargiu et al., 2000) and cultured in M199 medium supplemented with 2 mg bovine serum albumin, 3.6 mg Hepes, 100 U
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penicillin, 100 mg streptomycin/ml, 5% horse serum and 1 nmol/l insulin. Only cell suspensions with a viability (trypan blue exclusion test) of 80–85% or more were used. After 4 h the medium was changed to M199 as above, but without horse serum and with 10 nmol/l insulin, and incubated in a CO2 /air (5:95, v/v) atmosphere. The medium was changed daily. 2.3. Nitrites Nitrites and nitrates (after reduction) in the medium were measured with the Griess reagent (Gross, Jaffe, Levi, & Kilbourn, 1991). 2.4. Western blot analysis Total hepatocyte lysates were subjected to SDSPAGE on acrylamide gels. Proteins were electrophoretically transferred to PVDF membranes. Equal loading was confirmed by staining in Ponceau S solution. The blots were incubated with desired primary antibodies, then incubated with peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulins in TBSTween containing 2% (w/v) non-fat dry milk as previously described (Vargiu et al., 2000) and developed with the ECL reagents according to manufacturer’s instructions. In order to evaluate levels of GSK3 and IB-␣, hepatocyte lysates in RIPA buffer were immunoprecipitated with specific antibodies and processed with the standard Protein-A sepharose beads technique. To evaluate the degree of phosphorylation, blots were stripped and reprobed with anti-phospho-GSK-3 (1:1000) or with anti-phosphoIB-␣ (1:500).
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tion efficiency in our experiments was estimated to be approximately 80 %, as evaluated by analysis of gfp fluorescent cells. 2.6. Nuclear extracts Nuclear extracts were obtained by a slight modification of the procedure described by Lee, Bindereif, and Green (1988). Briefly, hepatocytes were harvested in hypotonic buffer (10 mM Hepes, 1.5 mM MgCl2 , 10 mM KCl, 0,5 mM DTT, 1 mM PMSF, pH 7.9), added with Triton X-100 (0.1% final concentration) and incubated for 15 min on ice. Cells were then disrupted by repeated passages into a syringe with a narrow-gauge (no. 27) needle and cells lysis was checked by observation under phase-contrast microscope. Nuclear pellets, obtained by centrifugation at 11,000 × g for 20 min, were rinsed once in hypotonic buffer, resuspended in hypertonic buffer (20 mM Hepes, 1.5 mM MgCl2 , 25% (v/v) glycerol, 420 mM NaCl, 0.5 mM DTT, 1 mM PSMF, 0.2 mM EDTA, pH 7.9), gently shaked for 30 min at 4 ◦ C and then centrifuged at 21,000 × g for 7 min. The final supernatant was processed for Western blot analysis as described. 2.7. Immunofluorescence microscopy Cultured hepatocytes were fixed in acetone/methanol (1:1, v/v) at −20 ◦ C, washed with PBS containing 0.1% Triton X-100, blocked in 1% bovine serum albumin and incubated with monoclonal anti--catenin (1:100) at 4 ◦ C overnight, followed by detection with anti-mouse Cy3-coniugated secondary antibody and nuclei counterstaining with Hoechst 33258.
2.5. Dominant negative IκB-α transient transfection
2.8. Statistical analysis
Primary hepatocytes were transiently transfected with 10 g of a plasmid encoding the dominant negative IB-␣ (IBDN, originally described by Whiteside et al. (1995), and kindly provided by Prof. C. Ponzetto, University of Torino) and 1 g of a plasmid encoding Green Fluorescent Protein (gfp) (Clontech) for monitoring transfection. Briefly 107 cells have been resuspended in 0.8 ml of Ca2+ - and Mg2+ -free phosphate buffered saline (PBS) solution with the above indicated amounts of constructs and electroporated with a Gene Pulser apparatus (BioRad, Milano, Italy) with the following settings: 200 V, 500 F. After pulse, cells were left 15 min at 4 ◦ C for recovery and then plated in complete growth medium for various times and subsequently harvested for assay as indicated elsewhere. Transfec-
Data in bar graphs represent means ± S.E.M. and means were obtained from average data of at least three independent experiments. Statistical analysis was performed by Student’s t-test (p < 0.05 was considered significant). 3. Results 3.1. Disruption of cell-to-cell contacts during hepatocyte isolation induces β-catenin nuclear translocation, followed by NF-κB involvement and iNOS induction Changes of -catenin subcellular localization in cultured rat hepatocytes were first investigated by indirect
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Fig. 1. Time-dependent changes in -catenin subcellular localization in cultured hepatocytes. Panel A: Indirect immunofluorescence staining for -catenin (left images, red fluorescence) in isolated rat hepatocytes at the indicated time points of culture after plating (primary antibody: monoclonal anti -catenin, 1:100 (v/v), final dilution; secondary antibody: anti-mouse Cy3-coniugated, 1:1000 (v/v), final dilution). Nuclei were counter-stained by using Hoechst 33258 fluorescent dye (center images, blue fluorescence). Electronic merging of images is also provided (right images). Original magnification 400×. Panel B: Nuclear extracts were obtained from freshly isolated hepatocytes (lane 1), or from hepatocytes after 4 (lane 2) and 24 h (lane 3) after plating. Western blot analysis was performed as described in Section 2. Luminograms and morphological images are representative of at least three experiments with similar results. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
immunofluorescence. As early as 4 h after seeding, catenin (Fig. 1A) was found in cytosplasmic and nuclear compartments; subsequently it disappeared from nuclei within 24 h to recover its classic plasma membrane localization within 48 h. Results were confirmed by Western blot analysis of nuclear extracts (Fig. 1B). These early changes were part of a scenario, already described in the literature (Loyer et al., 1996; Monga et al., 2003), consisting in activation of NF-B and increased synthesis of iNOS, as evaluated by Western blot analysis of nuclear levels of p65 subunit (Fig. 2A) and iNOS
protein levels (Fig. 2B), both peaking at 24 h after seeding. Maximal iNOS levels (24 h) were associated with the peak in NO production, as indicated by the accumulation of nitrites in the medium (Fig. 2C). It should be noted that activation of NF-B and increased synthesis of iNOS, that may potentially depend on different stimuli (oxidative stress, endotoxins in collagenase preparations, or proinflammatory cytokine’s release during isolation), were again a transient event since iNOS protein rapidly disappeared within 48–72 h (Fig. 2B).
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Fig. 2. Time-dependent changes of NF-B p65 subunit, iNOS protein levels and NO release in cultured hepatocytes. Panel A: Western blot analysis for NF-B p65 as performed on nuclear extracts obtained from freshly isolated hepatocytes (lane 1), or from hepatocytes after 4 (lane 2) and 24 h (lane 3) from plating. Panel B: Western blot analysis for iNOS as performed on total cell lysates obtained from freshly isolated hepatocytes (lane 1), or from hepatocytes after 4 (lane 2), 24 (lane 3), 48 (lane 4) and 72 h (lane 5) after plating. Luminograms (panels A and B) are representative of at least three experiments with similar results. Panel C: NO release by hepatocytes cultured for the indicated time points as evaluated in terms of nanomoles of nitrites/nitrates generated within 24 h by 2 × 105 cells. Data are presented as mean ± S.D. (n = 7). *p < 0.01 vs. data obtained at 72 h.
3.2. β-Catenin nuclear translocation and iNOS induction are reproduced in stabilized cultured hepatocytes exposed to EGTA/LiCl The early and transient changes described in the previous paragraph are likely to represent different features of the same “adaptative response” first elicited by disruption of cell–cell and cell–substrate interactions and then switched off by the progressive re-constitution of these interactions in culture. Since intracellular levels of free -catenin are regulated by the activity of GSK3, we performed experiments to evaluate in stabilized cultured hepatocytes (i.e. 48 h after seeding) whether a rise of intracellular -catenin, specifically obtained by EGTA-dependent disruption of cadherin-cadherin interactions in adherens junctions, and/or specific inactivation by LiCl of GSK-3 may result in a similar response. Exposure of stabilized hepatocytes to EGTA/LiCl for 2 h was followed by a rapid mobilization of -
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catenin resulting again in a significant increase of its cytoplasmic and nuclear levels (Fig. 3A and B). When EGTA/LiCl (mimicking Wnt--catenin pathway activation) (Orlandini, Semboloni, & Oliviero, 2003) was removed and replaced with fresh medium containing Ca2+ , a progressive reversion of -catenin cellular localization (disappearance of nuclear localization and appearance of -catenin at membrane level) was detected after 15 h (Fig. 3A and B) and completed within 24 h (data not shown). The inhibitory, Wnt-mimicking, action of LiCl, resulting in phosphorylation and inactivation of GSK3 (Zhang et al., 2003) was not reproduced by indirubin that is known to prevent ATP binding in GSK-3 active site (Meijer et al., 2003) without affecting the state of phosphorylation (Fig. 4). The efficacy of -catenin signaling was corroborated by increased protein levels of cyclin D1 and c-myc, two representative -catenin target – and cell cycle – related genes (Fig. 5 panels A and B); along these lines, it should be noted that LiCl can also stimulate hepatocyte proliferation, as already preliminarly reported (Compagnone et al., 2006, manuscript in preparation). Once assessed the experimental model (i.e. -catenin nuclear translocation elicited in stabilized hepatocytes by EGTA/LiCl) we investigated whether nuclear translocation of -catenin may be linked to up-regulation of iNOS expression. EGTA/LiCl and EGTA/indirubin (both inhibitors of GSK-3) were effective in increasing iNOS protein levels 24 h after stimulation (Fig. 6), as confirmed by analysis of nitrite’s accumulation in culture medium (3.2 nmol/24 h/20 × 105 cells vs. undetectable values in control cultures). 3.3. β-Catenin nuclear translocation and iNOS induction are mechanistically linked through β-Trcp1 induction and NF-κB activation We next performed experiments to identify the mechanism(s) linking -catenin nuclear translocation to iNOS induction after exposure to the stimulus represented by EGTA/LiCl. First, iNOS up-regulation was abolished by pre-treatment with dexamethasone, suggestive of an involvement of NF-B, or when LiCl was substituted with KCl, the latter being unable to inhibit GSK-3 (Fig. 6). The involvement of NFB by EGTA/LiCl-induced nuclear translocation of -catenin was also suggested by a very significant increase in p65 subunit nuclear levels (Fig. 7A) following exposure of stabilized cultured hepatocytes to EGTA/LiCl (15 h). Moreover, EGTA/LiCl also resulted in increased levels of phosphorylated IB-
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Fig. 3. Exposure of stabilized cultured hepatocytes to medium containing EGTA and LiCl induces a transient nuclear translocation of -catenin. Hepatocytes were plated and left in culture for 48 h, then either left untreated (control, CNT) or exposed for 2 h to a Wnt-mimicking medium containing 2 mM EGTA and 10–30 mM LiCl (EGTA/LiCl); at the end of this period medium was replaced and hepatocytes were exposed for 15 h to fresh normal medium to restore normal calcium concentration (Ca2+ 15 h). Panel A: Indirect immunofluorescence staining for -catenin (red fluorescence), nuclear counterstain (blue fluorescence) and electronic merging (right images) were performed as described in the legend of Fig. 1 for control cells (CNT), for cells exposed for 2 h to EGTA and 10 mM LiCl (EGTA/LiCl) and for cells exposed afterwards for 15 h to calcium containing fresh medium (Ca2+ 15 h). Original magnification 400×. Panel B: Western blot analysis for -catenin as performed on nuclear extracts that were obtained from cells submitted to the following experimental conditions: (1) untreated control (CNT) hepatocytes after 2 h; (2) hepatocytes exposed for 2 h to EGTA and 10 mM LiCl; (3) hepatocytes exposed for 2 h to EGTA and 30 mM LiCl; (4) untreated control (CNT) hepatocytes after additional 15 h; (5) and (6) hepatocytes first exposed for 2 h to EGTA and 10 mM LiCl (5) or to EGTA and 30 mM LiCl (6) and then both cultured for additional 15 h in normal medium containing calcium. Luminograms and morphological images are representative of at least three experiments with similar results. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
␣ and reduced levels of total IB-␣ (Fig. 7B and C). The latter event did not occur if cells were pretreated with dexamethasone or LiCl was replaced by KCl. The possible link between -catenin nuclear translocation and NF-B involvement was further suggested by EGTA/LiCl-dependent up-regulation of -TrCP1 protein, a well known target gene for -catenin/TCF/LEF, that recognize phosphorylated forms of -catenin and IB-␣ leading them to ubiquitination/proteasomal degradation (Fig. 8). Once again, up-regulation of -TrCP1
was completely abolished by replacing LiCl with KCl. Finally, in order to unequivocally assess the mechanistic link between changes in -catenin subcellular localization, NF-B activation and iNOS induction, experiments were performed on hepatocytes transfected or not with dominant/negative IB-␣ constructs. Cells expressing dominant/negative IB-␣ (Fig. 9) were completely unable to synthetize iNOS in response to either TNF (positive control) or to the EGTA/LiCl treatment, as usually detected in non-transfected cells.
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Fig. 4. Changes in the levels of phosphorylated GSK-3 in cultured hepatocytes. Hepatocytes were plated and stabilized for 48 h in culture, then either left untreated (control) or exposed for 2 h to EGTA/LiCl, EGTA/indirubin or EGTA/KCl (see later) and finally left for additional 15 h in fresh medium containing calcium. Western blot analysis was performed on total cell lysates, immunoprecipitated with a polyclonal antibody raised against total GSK-3 (panel A), that were obtained from either untreated control cells (lane 1), from cells treated with 2 mM EGTA and 20 M indirubin (lane 2), from cells treated with 2 mM EGTA, 30 mM LiCl (lane 3) or from cells treated with 2 mM EGTA, 30 mM KCl (lane 4). Membranes were stripped and reprobed with a polyclonal antibody directed against phosphorylated (ser9) GSK-3 (panel B). Luminograms are representative of at least three experiments with similar results.
4. Discussion
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Fig. 6. Changes of iNOS levels in cultured hepatocytes. Hepatocytes were plated and stabilized for 48 h in culture, then left untreated (control, lane 1) or exposed for 2 h to 2 mM EGTA and 20 M indirubin (lane 2), or 2 mM EGTA and 30 mM LiCl (lane 3), or 2 mM EGTA, 30 mM LiCl and 0.1 M dexamethasone (lane 4) or 2 mM EGTA and 30 mM KCl (lane 5) and finally left for additional 24 h in fresh medium containing calcium. Western blot analysis was performed on total cell lysates with monoclonal antibody raised against iNOS. Luminograms are representative of at least three experiments with similar results.
(Hortelano, Dewez, Genaro, Diaz-Guerra, & Bosca, 1995; Rai et al., 1998). The two-step collagenase method for isolation of hepatocytes can trigger their G0/G1 transition and induce several immediate–early proto-oncogenes having a crucial role in regulating cell cycle. Some of these genes (cyclin D1, c-myc, etc.) are known targets for the catenin/TCF/LEF complex. Here we detected changes of -catenin subcellular localization during the process of hepatocyte isolation and culture plating to investigate mechanistically whether these changes were related to
Hepatocytes “in vivo” are poorly responsive to growth factors and become competent only after a “priming” potentially induced by several pathophysiological conditions, including partial hepatectomy, necrotic injury, metabolic stress and more generally, conditions resulting in disruption of cell-to-cell contacts or alterations of extracellular matrix. Most of these conditions, involving hepatocyte’s compensatory hyperplastic response, are also commonly associated with induction of iNOS and NO generation, a pro-survival feature able to significantly prevent cell death mediated by different cytokines
Fig. 5. Changes in protein levels of -catenin target genes in cultured hepatocytes. Hepatocytes were plated and stabilized for 48 h in culture, then left untreated (control, lane 1) or exposed for 2 h to 2 mM EGTA and 30 mM LiCl (lane 2) or 2 mM EGTA and 30 mM KCl (lane 3) and finally left for additional 15 h in fresh medium containing calcium. Western blot analysis was performed on total cell lysates with monoclonal antibodies raised against cyclin D1 (panel A) or against c-Myc (panel B). Luminograms are representative of at least three experiments with similar results.
Fig. 7. Involvement of NF-B in EGTA/LiCl-induced events. Panel A: Hepatocytes were plated and stabilized for 48 h in culture, then left untreated (control, lane 1) or exposed for 2 h to 2 mM EGTA and 10 mM LiCl (lane 2) or 2 mM EGTA and 30 mM LiCl (lane 3) and finally left for additional 15 h in fresh medium containing calcium. Western blot analysis was performed on nuclear extract with specific monoclonal antibody against NF-B p65. Panel B: Cells were plated and stabilized for 48 h in culture, then left untreated (control, lane 1) or exposed for 2 h to 2 mM EGTA and 30 mM LiCl (lane 2), 2 mM EGTA, 30 mM LiCl and 0.1 M dexamethasone (lane 3) or 2 mM EGTA and 30 mM KCl (lane 4) and finally left for additional 15 h in fresh medium containing calcium. Western blot analysis was performed on total cell lysates, immunoprecipitated with a polyclonal antibody raised against IB-␣. Panel C: The same membranes of panel B were re-blotted with a monoclonal antibody directed against phosphorylated (ser32) IB␣. Luminograms are representative of at least three experiments with similar results.
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Fig. 8. -TrCP1 levels in hepatocytes cultures. Hepatocytes were plated and stabilized for 48 h in culture, then left untreated (control, lane 1) or exposed for 2 h to 2 mM EGTA and 30 mM LiCl (lane 2) or 2 mM EGTA and 30 mM KCl (lane 3) and finally left for additional 15 h in fresh medium containing calcium. Western blot analysis was performed on total cell lysates using a specific polyclonal antibody for TrCP1. Luminograms are representative of at least three experiments with similar results.
iNOS induction and NO generation in conditions mimicking early tissue disruption. As early as 4 h after disruption of adherens junctions by EGTA perfusion, -catenin was homogenously distributed in the cytoplasm and, in more than 90% of cells, translocated within nuclei. Although caution should be always used when referring data obtained in an “in vitro” experimental model to “in vivo” conditions, it should be noted that early nuclear translocation of -catenin observed in primary cultured hepatocytes resembled the homologous “in vivo” event that was carefully described in regenerating liver early after partial hepatectomy (Monga, Pediaditakis, Mule, Stolz, & Michalopoulos, 2001). Within the next 36–40 h of culture, -catenin was back to its physiological plasma membrane localization, by binding the cytosolic domain of E-cadherin. Within the same time-frame, -catenin nuclear translocation was followed by transient activation of NF-B and iNOS induction. According to Paine and Andreakos (2004), the main stimulus responsible for NF-B activation and iNOS induction in our specific experimental conditions should be represented by oxidative stress
Fig. 9. iNOS induction in cells treated with the Wnt-mimicking stimulus is dependent on NF-B. Freshly isolated hepatocytes were either transfected (lanes 3 and 5) or not with a plasmid encoding for dominant negative IB-␣ (IBDN) and a plasmid encoding for Green Fluorescent Protein (gfp) to monitor transfection efficiency. All cells were then plated and stabilized in culture for 48 h and then left untreated (lane 1) or exposed for 24 h to TNF (200 ng/ml) (lanes 2 and 3) or to EGTA/LiCl 30 mM for 2 h and then finally left for additional 15 h in fresh medium containing calcium (lanes 4 and 5). Luminograms are representative of two experiments with similar results.
that was likely to occur during cell isolation and culture plating, although the involvement of other stimuli, including lipotheicoic acid contamination of collagenase or release of pro-inflammatory cytokines, cannot be ruled out. In order to exclude all these stimuli and mechanistically investigate relationships between activation of -catenin transcriptional activity and NF-B-dependent iNOS induction, we decided to wait until 48 h (i.e. a time point in which -catenin was back to its physiological subcellular localization and iNOS expression down-regulated) before treating cultured hepatocytes with stimuli able to re-activate -catenin pathway. As soon as 2 h after treatment with EGTA and LiCl, the latter used to prevent -catenin phosphorylation and degradation, -catenin left hepatocyte membranes to enter cytoplasm and nuclear compartments. -Catenin nuclear translocation, in turn, was once again followed by nuclear translocation of NF-B, iNOS induction and nitrite accumulation in culture medium. iNOS induction, in particular, was strictly related to inhibition of GSK-3, as confirmed by data obtained with both LiCl and indirubin, but it was abolished by concomitant treatment with dexamethasone, a drug able to prevent cytoplasmic depletion of IB-␣ without affecting its state of phosphorylation (De Vera et al., 1997). The mechanistic relationships with NF-B activation was unequivocally shown by lack of iNOS induction by EGTA/LiCl in hepatocytes expressing dominant/negative IB-␣. An overall analysis of our data then suggests that adherens junctions disassembly in rat hepatocyte, a potential major determinant of hepatocyte “priming” for proliferation and survival in pathophysiological conditions, may operate by triggering an early and transient activation of -catenin pathway that, in turn, can result in a coordinated and sequential increase in -TrCP1 expression and NF-B-dependent iNOS induction (Fig. 10). This study provides novel evidence for NF-B activation as a necessary step linking adherens junctions disruption and Wnt/-catenin signaling to iNOS upregulation in hepatocytes. The functional relationship between NF-B and GSK-3 has been a matter of debate in the last few years, with some reports suggesting a positive link and others suggesting exactly the opposite scenario (i.e. GSK-3 activity as necessary for NF-B activation). In the only published study on cultured rat hepatocytes, Schwabe and Brenner (2002) analysed the experimental problem of TNF␣-induced apoptosis (a different approach to our present one). They described that inhibition of GSK-3 by LiCl resulted in an increased sensitivity to TNF␣-induced apoptosis,
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Fig. 10. Schematic representation of major signaling events connecting inhibition of GSK-3 and resulting -catenin nuclear translocation to increased transcription of TrCp1 that, in turn, leads to NF-B-dependent iNOS induction (see text for more details).
suggesting that GSK-3 inhibition was related to downregulation of NF-B dependent gene iNOS. However, this reported “indirect relationships” between GSK-3 and NF-B deserves some comments. First, in their experimental model the driving force for iNOS induction was TNF␣ itself, which is known to also activate NF-B. As a matter of fact, as authors clearly underlined, that study was reporting a down regulation of TNF␣-dependent event but, surprisingly, inhibition of GSK-3 by LiCl was not followed neither by inhibition of TNF␣-dependent NF-B DNA binding nor alteration in IB-␣ degradation, IB kinase activity or p65 nuclear import/export. Surprisingly, no changes in NF-B related parameters have been documented in the presence of LiCl alone; moreover, these results were puzzling since -catenin signaling activation through GSK-3 inhibition usually operates as a survival event (Calvisi, Ladu, Factor, & Thorgeirsson, 2004; Emanuele et al., 2004; Hahn et al., 2006; Sinha et al., 2005). Our data, however, are in agreement with a number of more direct literature findings obtained in different experimental models or cell lines. Wang, Adhikari, Li, Guan, and Hall (2004) have described that upregulation of -TrCp1 can accelerate the rate of IB-␣ degradation, leading to increased NF-B activity in vascular smooth muscle cells. Along these lines, Bournat, Brown,
and Soler (2000) described that activation of Wnt/catenin signaling pathway in PC12 cells resulted in NF-B activation, a condition that has been conceptually reproduced by others using LiCl to inhibit GSK-3 in human intestinal epithelial cells (Nemeth et al., 2002) or in renal medullary interstitial cells (Rao, Hao, & Breyer, 2004). Even more important, a direct in vivo relationship between -catenin and iNOS induction has been very recently reported (Du et al., 2006) in which hepatic levels of both iNOS and nuclear -catenin were found to be significantly increased after administration of LiCl. Similarly, in a non-hepatic “in vivo” experimental model, adherens junction disruption was again followed by the sequence of -catenin activation and increased expression of iNOS (Lee, Mruk, Wong, & Cheng, 2005). Acknowledgements Financial support was from Regione Piemonte and Fondazione CRT (Torino, MP), University of Torino (MP, SC) and MIUR (Rome, PRIN project, SC). References Barth, A. I., Nathke, I. S., & Nelson, W. J. (1997). Cadherins, catenins and APC protein: interplay between cytoskeletal complexes and signaling pathways. Curr. Opin. Cell. Biol., 9, 683–690.
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The International Journal of Biochemistry & Cell Biology 40 (2008) 1861–1871
-Catenin triggers nuclear factor B-dependent up-regulation of hepatocyte inducible nitric oxide synthase Andrea Bandino, Alessandra Compagnone, Vittoria Bravoco, Carlo Cravanzola, Anna Lomartire, Chiara Rossetto, Erica Novo, Stefania Cannito, Lorenzo Valfr`e di Bonzo, Elena Zamara, Riccardo Autelli, Maurizio Parola, Sebastiano Colombatto ∗ Dipartimento di Medicina e Oncologia Sperimentale, University of Torino, Torino, Italy Received 21 November 2007; received in revised form 18 January 2008; accepted 21 January 2008 Available online 7 February 2008
Abstract Disruption of cell-to-cell contacts, as observed in many pathophysiological conditions, prime hepatocytes for compensatory hyperplastic response that involves induction of several genes, including proto-oncogenes and other gene targets of -catenin signaling pathway. By using cultured hepatocytes and experimental models of adherens junction disruption we have investigated changes in -catenin subcellular localization and their relationships with inducible nitric oxide synthase (iNOS) expression. Two experimental models were employed: (a) rat hepatocytes obtained by collagenase liver perfusion within the first 48 h of culture; (b) 48-h old cultured hepatocytes, transiently transfected or not with a plasmid encoding for dominant/negative inhibitory kappa B␣, exposed to ethylene glycol-bis-(2-aminoethylether)-N,N,N ,N -tetraacetic acid/LiCl treatment. -Catenin signaling and cellular localization, iNOS expression and nuclear factor B involvement, were investigated using morphological, cell and molecular biology techniques. E-cadherin-mediated disruption of cell-to-cell contacts induces early -catenin translocation from membrane to cytoplasm and nuclear compartments, events that are followed by up-regulation of c-myc, cyclin D1 and -transducin repeatcontaining protein expression. This, in turn, resulted eventually in iNOS induction that was mechanistically related to nuclear factor B activation, as unequivocally shown in cells expressing dominant negative inhibitory kappa B-␣. Our data indicate that E-cadherin disassembly and concomitant inactivation of glycogen synthase kinase-3 result in nuclear factor B-dependent induction of iNOS in hepatocytes. © 2008 Published by Elsevier Ltd. Keywords: Lithium; -TrCp1; IB-␣; -Catenin
1. Introduction -Catenin is a multifunctional protein involved mainly in cadherin-mediated cell–cell adhesion (Barth, ∗
Corresponding author at: Dipartimento di Medicina e Oncologia Sperimentale, Sezione di Biochimica, Via Michelangelo 27, 10126 Torino, Italy. Tel.: +39 011 670 5308; fax: +39 011 670 5311. E-mail address: [email protected] (S. Colombatto). 1357-2725/$ – see front matter © 2008 Published by Elsevier Ltd. doi:10.1016/j.biocel.2008.01.029
Nathke, & Nelson, 1997) and in the control of growth and differentiation of several tissues during foetal and adult life. -Catenin, once detached from cytoplasmic domain of E-cadherins, can act as intracellular signal intermediate of the Wnt/wingless pathway (Cadigan & Nusse, 1997; Polakis, 2000) in central nervous system, epidermis (Huelsken, Vogel, Erdmann, Cotsarelis, & Birchmeier, 2001), intestine (Peifer, 2002) and liver (Monga et al., 2003).
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In unstimulated cells, cytoplasmic levels of free -catenin are negatively controlled by means of ubiquitination and subsequent degradation by the 26S proteasome. Glycogen synthase kinase-3 (GSK-3), which is active when complexed with axin, casein kinase 1 (CK1) and the adenomatous polyposis coli gene product (APC), plays a crucial role in ubiquitination by phosphorylating -catenin on 41-threonine residue and multiple serine residues (Ser 33, 37, 61). Activation of the Wnt signal transduction pathway results from the binding of one of the several known components of Wnt-family of extracellular proteins to the receptors Frizzled/LPR5/6. This promotes the disruption of the complex GSK-3–axin–CK1–APC, prevents -catenin phosphorylation and degradation, resulting in increased cytoplasmic levels of -catenin that, in turn, translocates into the nucleus to bind factors TCF/LEF and promote their activation. The latter complex induces transcription of several target genes, including c-myc (He et al., 1998), cyclin D1 (Shtutman et al., 1999), c-jun, fra-1, uPAR (Mann et al., 1999), CD44 (Wielenga et al., 1999), VEGF (Zhang, Phiel, Spece, Gurvich, & Klein, 2003), fibronectin (Gradl, K¨uhl, & Wedlich, 1999), cyclooxygenase-2 (Haertel-Wiesmann, Liang, Fantl, & Williams, 2000) and -transducin repeat-containing protein (-TrCp1) (Spiegelman et al., 2000). -TrCp1, a F-box protein component of a SKp1/Cul1/F-box-type ubiquitin ligase complex able to bind phosphorylated and unphosphorylated target molecules, selectively recognizes only phosphorylated forms of the nuclear factor kappa B (NF-B) inhibitor (IB-␣) and -catenin (Nakayama et al., 2003). -TrCp1 gene expression is then controlled by -catenin (Spiegelman et al., 2000) and it represents a direct link between -catenin and NF-B activation. Primary cultures of rat hepatocytes are a reliable experimental model widely used for studies of tissue engineering, metabolic pathways and signal transduction. Isolation procedure requires ethylene glycol-bis-(2-aminoethylether)-N,N,N ,N -tetraacetic acid (EGTA)/collagenase-dependent disgregation of liver parenchyma and also disrupts cell–cell interactions, rapidly recovered by hepatocytes within 24/36 h of primary culture. This procedure can effectively trigger G0/G1 transition and induce cell cycle-dependent immediate–early proto-oncogenes, then mimicking what is likely to occur during a number of pathophysiological conditions, including partial hepatectomy, necrotic injury, metabolic stress and, more generally, conditions resulting in disruption of cell-to-cell contacts or alterations of extracellular matrix (Etienne et al., 1988; Ikeda, Sawada, Fujinaga, Minase, & Mori, 1989;
Liu, Mars, Zarnegar, & Michalopoulos, 1994; Sawada, 1989). Freshly isolated – and early cultured – hepatocytes have been reported, even in the absence of mitogens, to undergo up-regulation of immediate/early oncogenes (Loyer et al., 1996), most of them being targets of the -catenin/TCF/LEF complex, as well as of inducible nitric oxide synthase (iNOS). iNOS induction, which is a common finding in pathophysiological conditions, is prominent and transient during the first 24 h of culture and leads to accumulation of nitrites in culture medium (Pittner & Spitzer, 1993; Vargiu, Belliardo, Cravanzola, Grillo, & Colombatto, 2000; Vernia, Beaune, Coloma, & Lopez-Garcia, 2001). Moreover, iNOS may be upregulated by isolation-related stimuli like oxidative stress, endotoxins (LPS or lipotheicoic acid) in collagenase preparations and/or release of proinflammatory cytokines (Paine & Andreakos, 2004). In the present study we report that both early cultured hepatocytes as well as stabilized cells treated with the Wnt-mimicking stimulus EGTA/LiCl, used as models of disruption of cell-to-cell contacts, undergo a sequence of events that mechanistically links -catenin nuclear translocation to iNOS induction through NF-B activation. 2. Materials and methods 2.1. Materials Peroxidase- and Cy3-coniugated secondary antibodies were from Amersham (Little Chalfont, Buckinghamshire, UK), horse serum from SPA (Milan, Italy), collagenase, penicillin/streptomycin, M199, insulin, dexamethasone, indirubin-3 -oxime, LiCl, phenylmethylsulfonyl fluoride, Hoechst 33258 and all the other chemicals from Sigma Chemical Co. (St. Louis, MO, USA). All antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) except anti-NOS II (Transduction Laboratories, Lexington, KY, USA) and anti-phospho-GSK-3 (Cell Signaling Technology Inc., Danvers, MA, USA). 2.2. Experimental animals Hepatocytes were isolated by means of collagenase/ perfusion method from male Wistar rats (150–200 g) that were used according to national and local guidelines. Hepatocytes were plated (7 × 104 cells/cm2 ) on culture dishes coated with rat tail tendon collagen (Vargiu et al., 2000) and cultured in M199 medium supplemented with 2 mg bovine serum albumin, 3.6 mg Hepes, 100 U
A. Bandino et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 1861–1871
penicillin, 100 mg streptomycin/ml, 5% horse serum and 1 nmol/l insulin. Only cell suspensions with a viability (trypan blue exclusion test) of 80–85% or more were used. After 4 h the medium was changed to M199 as above, but without horse serum and with 10 nmol/l insulin, and incubated in a CO2 /air (5:95, v/v) atmosphere. The medium was changed daily. 2.3. Nitrites Nitrites and nitrates (after reduction) in the medium were measured with the Griess reagent (Gross, Jaffe, Levi, & Kilbourn, 1991). 2.4. Western blot analysis Total hepatocyte lysates were subjected to SDSPAGE on acrylamide gels. Proteins were electrophoretically transferred to PVDF membranes. Equal loading was confirmed by staining in Ponceau S solution. The blots were incubated with desired primary antibodies, then incubated with peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulins in TBSTween containing 2% (w/v) non-fat dry milk as previously described (Vargiu et al., 2000) and developed with the ECL reagents according to manufacturer’s instructions. In order to evaluate levels of GSK3 and IB-␣, hepatocyte lysates in RIPA buffer were immunoprecipitated with specific antibodies and processed with the standard Protein-A sepharose beads technique. To evaluate the degree of phosphorylation, blots were stripped and reprobed with anti-phospho-GSK-3 (1:1000) or with anti-phosphoIB-␣ (1:500).
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tion efficiency in our experiments was estimated to be approximately 80 %, as evaluated by analysis of gfp fluorescent cells. 2.6. Nuclear extracts Nuclear extracts were obtained by a slight modification of the procedure described by Lee, Bindereif, and Green (1988). Briefly, hepatocytes were harvested in hypotonic buffer (10 mM Hepes, 1.5 mM MgCl2 , 10 mM KCl, 0,5 mM DTT, 1 mM PMSF, pH 7.9), added with Triton X-100 (0.1% final concentration) and incubated for 15 min on ice. Cells were then disrupted by repeated passages into a syringe with a narrow-gauge (no. 27) needle and cells lysis was checked by observation under phase-contrast microscope. Nuclear pellets, obtained by centrifugation at 11,000 × g for 20 min, were rinsed once in hypotonic buffer, resuspended in hypertonic buffer (20 mM Hepes, 1.5 mM MgCl2 , 25% (v/v) glycerol, 420 mM NaCl, 0.5 mM DTT, 1 mM PSMF, 0.2 mM EDTA, pH 7.9), gently shaked for 30 min at 4 ◦ C and then centrifuged at 21,000 × g for 7 min. The final supernatant was processed for Western blot analysis as described. 2.7. Immunofluorescence microscopy Cultured hepatocytes were fixed in acetone/methanol (1:1, v/v) at −20 ◦ C, washed with PBS containing 0.1% Triton X-100, blocked in 1% bovine serum albumin and incubated with monoclonal anti--catenin (1:100) at 4 ◦ C overnight, followed by detection with anti-mouse Cy3-coniugated secondary antibody and nuclei counterstaining with Hoechst 33258.
2.5. Dominant negative IκB-α transient transfection
2.8. Statistical analysis
Primary hepatocytes were transiently transfected with 10 g of a plasmid encoding the dominant negative IB-␣ (IBDN, originally described by Whiteside et al. (1995), and kindly provided by Prof. C. Ponzetto, University of Torino) and 1 g of a plasmid encoding Green Fluorescent Protein (gfp) (Clontech) for monitoring transfection. Briefly 107 cells have been resuspended in 0.8 ml of Ca2+ - and Mg2+ -free phosphate buffered saline (PBS) solution with the above indicated amounts of constructs and electroporated with a Gene Pulser apparatus (BioRad, Milano, Italy) with the following settings: 200 V, 500 F. After pulse, cells were left 15 min at 4 ◦ C for recovery and then plated in complete growth medium for various times and subsequently harvested for assay as indicated elsewhere. Transfec-
Data in bar graphs represent means ± S.E.M. and means were obtained from average data of at least three independent experiments. Statistical analysis was performed by Student’s t-test (p < 0.05 was considered significant). 3. Results 3.1. Disruption of cell-to-cell contacts during hepatocyte isolation induces β-catenin nuclear translocation, followed by NF-κB involvement and iNOS induction Changes of -catenin subcellular localization in cultured rat hepatocytes were first investigated by indirect
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Fig. 1. Time-dependent changes in -catenin subcellular localization in cultured hepatocytes. Panel A: Indirect immunofluorescence staining for -catenin (left images, red fluorescence) in isolated rat hepatocytes at the indicated time points of culture after plating (primary antibody: monoclonal anti -catenin, 1:100 (v/v), final dilution; secondary antibody: anti-mouse Cy3-coniugated, 1:1000 (v/v), final dilution). Nuclei were counter-stained by using Hoechst 33258 fluorescent dye (center images, blue fluorescence). Electronic merging of images is also provided (right images). Original magnification 400×. Panel B: Nuclear extracts were obtained from freshly isolated hepatocytes (lane 1), or from hepatocytes after 4 (lane 2) and 24 h (lane 3) after plating. Western blot analysis was performed as described in Section 2. Luminograms and morphological images are representative of at least three experiments with similar results. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
immunofluorescence. As early as 4 h after seeding, catenin (Fig. 1A) was found in cytosplasmic and nuclear compartments; subsequently it disappeared from nuclei within 24 h to recover its classic plasma membrane localization within 48 h. Results were confirmed by Western blot analysis of nuclear extracts (Fig. 1B). These early changes were part of a scenario, already described in the literature (Loyer et al., 1996; Monga et al., 2003), consisting in activation of NF-B and increased synthesis of iNOS, as evaluated by Western blot analysis of nuclear levels of p65 subunit (Fig. 2A) and iNOS
protein levels (Fig. 2B), both peaking at 24 h after seeding. Maximal iNOS levels (24 h) were associated with the peak in NO production, as indicated by the accumulation of nitrites in the medium (Fig. 2C). It should be noted that activation of NF-B and increased synthesis of iNOS, that may potentially depend on different stimuli (oxidative stress, endotoxins in collagenase preparations, or proinflammatory cytokine’s release during isolation), were again a transient event since iNOS protein rapidly disappeared within 48–72 h (Fig. 2B).
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Fig. 2. Time-dependent changes of NF-B p65 subunit, iNOS protein levels and NO release in cultured hepatocytes. Panel A: Western blot analysis for NF-B p65 as performed on nuclear extracts obtained from freshly isolated hepatocytes (lane 1), or from hepatocytes after 4 (lane 2) and 24 h (lane 3) from plating. Panel B: Western blot analysis for iNOS as performed on total cell lysates obtained from freshly isolated hepatocytes (lane 1), or from hepatocytes after 4 (lane 2), 24 (lane 3), 48 (lane 4) and 72 h (lane 5) after plating. Luminograms (panels A and B) are representative of at least three experiments with similar results. Panel C: NO release by hepatocytes cultured for the indicated time points as evaluated in terms of nanomoles of nitrites/nitrates generated within 24 h by 2 × 105 cells. Data are presented as mean ± S.D. (n = 7). *p < 0.01 vs. data obtained at 72 h.
3.2. β-Catenin nuclear translocation and iNOS induction are reproduced in stabilized cultured hepatocytes exposed to EGTA/LiCl The early and transient changes described in the previous paragraph are likely to represent different features of the same “adaptative response” first elicited by disruption of cell–cell and cell–substrate interactions and then switched off by the progressive re-constitution of these interactions in culture. Since intracellular levels of free -catenin are regulated by the activity of GSK3, we performed experiments to evaluate in stabilized cultured hepatocytes (i.e. 48 h after seeding) whether a rise of intracellular -catenin, specifically obtained by EGTA-dependent disruption of cadherin-cadherin interactions in adherens junctions, and/or specific inactivation by LiCl of GSK-3 may result in a similar response. Exposure of stabilized hepatocytes to EGTA/LiCl for 2 h was followed by a rapid mobilization of -
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catenin resulting again in a significant increase of its cytoplasmic and nuclear levels (Fig. 3A and B). When EGTA/LiCl (mimicking Wnt--catenin pathway activation) (Orlandini, Semboloni, & Oliviero, 2003) was removed and replaced with fresh medium containing Ca2+ , a progressive reversion of -catenin cellular localization (disappearance of nuclear localization and appearance of -catenin at membrane level) was detected after 15 h (Fig. 3A and B) and completed within 24 h (data not shown). The inhibitory, Wnt-mimicking, action of LiCl, resulting in phosphorylation and inactivation of GSK3 (Zhang et al., 2003) was not reproduced by indirubin that is known to prevent ATP binding in GSK-3 active site (Meijer et al., 2003) without affecting the state of phosphorylation (Fig. 4). The efficacy of -catenin signaling was corroborated by increased protein levels of cyclin D1 and c-myc, two representative -catenin target – and cell cycle – related genes (Fig. 5 panels A and B); along these lines, it should be noted that LiCl can also stimulate hepatocyte proliferation, as already preliminarly reported (Compagnone et al., 2006, manuscript in preparation). Once assessed the experimental model (i.e. -catenin nuclear translocation elicited in stabilized hepatocytes by EGTA/LiCl) we investigated whether nuclear translocation of -catenin may be linked to up-regulation of iNOS expression. EGTA/LiCl and EGTA/indirubin (both inhibitors of GSK-3) were effective in increasing iNOS protein levels 24 h after stimulation (Fig. 6), as confirmed by analysis of nitrite’s accumulation in culture medium (3.2 nmol/24 h/20 × 105 cells vs. undetectable values in control cultures). 3.3. β-Catenin nuclear translocation and iNOS induction are mechanistically linked through β-Trcp1 induction and NF-κB activation We next performed experiments to identify the mechanism(s) linking -catenin nuclear translocation to iNOS induction after exposure to the stimulus represented by EGTA/LiCl. First, iNOS up-regulation was abolished by pre-treatment with dexamethasone, suggestive of an involvement of NF-B, or when LiCl was substituted with KCl, the latter being unable to inhibit GSK-3 (Fig. 6). The involvement of NFB by EGTA/LiCl-induced nuclear translocation of -catenin was also suggested by a very significant increase in p65 subunit nuclear levels (Fig. 7A) following exposure of stabilized cultured hepatocytes to EGTA/LiCl (15 h). Moreover, EGTA/LiCl also resulted in increased levels of phosphorylated IB-
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Fig. 3. Exposure of stabilized cultured hepatocytes to medium containing EGTA and LiCl induces a transient nuclear translocation of -catenin. Hepatocytes were plated and left in culture for 48 h, then either left untreated (control, CNT) or exposed for 2 h to a Wnt-mimicking medium containing 2 mM EGTA and 10–30 mM LiCl (EGTA/LiCl); at the end of this period medium was replaced and hepatocytes were exposed for 15 h to fresh normal medium to restore normal calcium concentration (Ca2+ 15 h). Panel A: Indirect immunofluorescence staining for -catenin (red fluorescence), nuclear counterstain (blue fluorescence) and electronic merging (right images) were performed as described in the legend of Fig. 1 for control cells (CNT), for cells exposed for 2 h to EGTA and 10 mM LiCl (EGTA/LiCl) and for cells exposed afterwards for 15 h to calcium containing fresh medium (Ca2+ 15 h). Original magnification 400×. Panel B: Western blot analysis for -catenin as performed on nuclear extracts that were obtained from cells submitted to the following experimental conditions: (1) untreated control (CNT) hepatocytes after 2 h; (2) hepatocytes exposed for 2 h to EGTA and 10 mM LiCl; (3) hepatocytes exposed for 2 h to EGTA and 30 mM LiCl; (4) untreated control (CNT) hepatocytes after additional 15 h; (5) and (6) hepatocytes first exposed for 2 h to EGTA and 10 mM LiCl (5) or to EGTA and 30 mM LiCl (6) and then both cultured for additional 15 h in normal medium containing calcium. Luminograms and morphological images are representative of at least three experiments with similar results. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
␣ and reduced levels of total IB-␣ (Fig. 7B and C). The latter event did not occur if cells were pretreated with dexamethasone or LiCl was replaced by KCl. The possible link between -catenin nuclear translocation and NF-B involvement was further suggested by EGTA/LiCl-dependent up-regulation of -TrCP1 protein, a well known target gene for -catenin/TCF/LEF, that recognize phosphorylated forms of -catenin and IB-␣ leading them to ubiquitination/proteasomal degradation (Fig. 8). Once again, up-regulation of -TrCP1
was completely abolished by replacing LiCl with KCl. Finally, in order to unequivocally assess the mechanistic link between changes in -catenin subcellular localization, NF-B activation and iNOS induction, experiments were performed on hepatocytes transfected or not with dominant/negative IB-␣ constructs. Cells expressing dominant/negative IB-␣ (Fig. 9) were completely unable to synthetize iNOS in response to either TNF (positive control) or to the EGTA/LiCl treatment, as usually detected in non-transfected cells.
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Fig. 4. Changes in the levels of phosphorylated GSK-3 in cultured hepatocytes. Hepatocytes were plated and stabilized for 48 h in culture, then either left untreated (control) or exposed for 2 h to EGTA/LiCl, EGTA/indirubin or EGTA/KCl (see later) and finally left for additional 15 h in fresh medium containing calcium. Western blot analysis was performed on total cell lysates, immunoprecipitated with a polyclonal antibody raised against total GSK-3 (panel A), that were obtained from either untreated control cells (lane 1), from cells treated with 2 mM EGTA and 20 M indirubin (lane 2), from cells treated with 2 mM EGTA, 30 mM LiCl (lane 3) or from cells treated with 2 mM EGTA, 30 mM KCl (lane 4). Membranes were stripped and reprobed with a polyclonal antibody directed against phosphorylated (ser9) GSK-3 (panel B). Luminograms are representative of at least three experiments with similar results.
4. Discussion
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Fig. 6. Changes of iNOS levels in cultured hepatocytes. Hepatocytes were plated and stabilized for 48 h in culture, then left untreated (control, lane 1) or exposed for 2 h to 2 mM EGTA and 20 M indirubin (lane 2), or 2 mM EGTA and 30 mM LiCl (lane 3), or 2 mM EGTA, 30 mM LiCl and 0.1 M dexamethasone (lane 4) or 2 mM EGTA and 30 mM KCl (lane 5) and finally left for additional 24 h in fresh medium containing calcium. Western blot analysis was performed on total cell lysates with monoclonal antibody raised against iNOS. Luminograms are representative of at least three experiments with similar results.
(Hortelano, Dewez, Genaro, Diaz-Guerra, & Bosca, 1995; Rai et al., 1998). The two-step collagenase method for isolation of hepatocytes can trigger their G0/G1 transition and induce several immediate–early proto-oncogenes having a crucial role in regulating cell cycle. Some of these genes (cyclin D1, c-myc, etc.) are known targets for the catenin/TCF/LEF complex. Here we detected changes of -catenin subcellular localization during the process of hepatocyte isolation and culture plating to investigate mechanistically whether these changes were related to
Hepatocytes “in vivo” are poorly responsive to growth factors and become competent only after a “priming” potentially induced by several pathophysiological conditions, including partial hepatectomy, necrotic injury, metabolic stress and more generally, conditions resulting in disruption of cell-to-cell contacts or alterations of extracellular matrix. Most of these conditions, involving hepatocyte’s compensatory hyperplastic response, are also commonly associated with induction of iNOS and NO generation, a pro-survival feature able to significantly prevent cell death mediated by different cytokines
Fig. 5. Changes in protein levels of -catenin target genes in cultured hepatocytes. Hepatocytes were plated and stabilized for 48 h in culture, then left untreated (control, lane 1) or exposed for 2 h to 2 mM EGTA and 30 mM LiCl (lane 2) or 2 mM EGTA and 30 mM KCl (lane 3) and finally left for additional 15 h in fresh medium containing calcium. Western blot analysis was performed on total cell lysates with monoclonal antibodies raised against cyclin D1 (panel A) or against c-Myc (panel B). Luminograms are representative of at least three experiments with similar results.
Fig. 7. Involvement of NF-B in EGTA/LiCl-induced events. Panel A: Hepatocytes were plated and stabilized for 48 h in culture, then left untreated (control, lane 1) or exposed for 2 h to 2 mM EGTA and 10 mM LiCl (lane 2) or 2 mM EGTA and 30 mM LiCl (lane 3) and finally left for additional 15 h in fresh medium containing calcium. Western blot analysis was performed on nuclear extract with specific monoclonal antibody against NF-B p65. Panel B: Cells were plated and stabilized for 48 h in culture, then left untreated (control, lane 1) or exposed for 2 h to 2 mM EGTA and 30 mM LiCl (lane 2), 2 mM EGTA, 30 mM LiCl and 0.1 M dexamethasone (lane 3) or 2 mM EGTA and 30 mM KCl (lane 4) and finally left for additional 15 h in fresh medium containing calcium. Western blot analysis was performed on total cell lysates, immunoprecipitated with a polyclonal antibody raised against IB-␣. Panel C: The same membranes of panel B were re-blotted with a monoclonal antibody directed against phosphorylated (ser32) IB␣. Luminograms are representative of at least three experiments with similar results.
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Fig. 8. -TrCP1 levels in hepatocytes cultures. Hepatocytes were plated and stabilized for 48 h in culture, then left untreated (control, lane 1) or exposed for 2 h to 2 mM EGTA and 30 mM LiCl (lane 2) or 2 mM EGTA and 30 mM KCl (lane 3) and finally left for additional 15 h in fresh medium containing calcium. Western blot analysis was performed on total cell lysates using a specific polyclonal antibody for TrCP1. Luminograms are representative of at least three experiments with similar results.
iNOS induction and NO generation in conditions mimicking early tissue disruption. As early as 4 h after disruption of adherens junctions by EGTA perfusion, -catenin was homogenously distributed in the cytoplasm and, in more than 90% of cells, translocated within nuclei. Although caution should be always used when referring data obtained in an “in vitro” experimental model to “in vivo” conditions, it should be noted that early nuclear translocation of -catenin observed in primary cultured hepatocytes resembled the homologous “in vivo” event that was carefully described in regenerating liver early after partial hepatectomy (Monga, Pediaditakis, Mule, Stolz, & Michalopoulos, 2001). Within the next 36–40 h of culture, -catenin was back to its physiological plasma membrane localization, by binding the cytosolic domain of E-cadherin. Within the same time-frame, -catenin nuclear translocation was followed by transient activation of NF-B and iNOS induction. According to Paine and Andreakos (2004), the main stimulus responsible for NF-B activation and iNOS induction in our specific experimental conditions should be represented by oxidative stress
Fig. 9. iNOS induction in cells treated with the Wnt-mimicking stimulus is dependent on NF-B. Freshly isolated hepatocytes were either transfected (lanes 3 and 5) or not with a plasmid encoding for dominant negative IB-␣ (IBDN) and a plasmid encoding for Green Fluorescent Protein (gfp) to monitor transfection efficiency. All cells were then plated and stabilized in culture for 48 h and then left untreated (lane 1) or exposed for 24 h to TNF (200 ng/ml) (lanes 2 and 3) or to EGTA/LiCl 30 mM for 2 h and then finally left for additional 15 h in fresh medium containing calcium (lanes 4 and 5). Luminograms are representative of two experiments with similar results.
that was likely to occur during cell isolation and culture plating, although the involvement of other stimuli, including lipotheicoic acid contamination of collagenase or release of pro-inflammatory cytokines, cannot be ruled out. In order to exclude all these stimuli and mechanistically investigate relationships between activation of -catenin transcriptional activity and NF-B-dependent iNOS induction, we decided to wait until 48 h (i.e. a time point in which -catenin was back to its physiological subcellular localization and iNOS expression down-regulated) before treating cultured hepatocytes with stimuli able to re-activate -catenin pathway. As soon as 2 h after treatment with EGTA and LiCl, the latter used to prevent -catenin phosphorylation and degradation, -catenin left hepatocyte membranes to enter cytoplasm and nuclear compartments. -Catenin nuclear translocation, in turn, was once again followed by nuclear translocation of NF-B, iNOS induction and nitrite accumulation in culture medium. iNOS induction, in particular, was strictly related to inhibition of GSK-3, as confirmed by data obtained with both LiCl and indirubin, but it was abolished by concomitant treatment with dexamethasone, a drug able to prevent cytoplasmic depletion of IB-␣ without affecting its state of phosphorylation (De Vera et al., 1997). The mechanistic relationships with NF-B activation was unequivocally shown by lack of iNOS induction by EGTA/LiCl in hepatocytes expressing dominant/negative IB-␣. An overall analysis of our data then suggests that adherens junctions disassembly in rat hepatocyte, a potential major determinant of hepatocyte “priming” for proliferation and survival in pathophysiological conditions, may operate by triggering an early and transient activation of -catenin pathway that, in turn, can result in a coordinated and sequential increase in -TrCP1 expression and NF-B-dependent iNOS induction (Fig. 10). This study provides novel evidence for NF-B activation as a necessary step linking adherens junctions disruption and Wnt/-catenin signaling to iNOS upregulation in hepatocytes. The functional relationship between NF-B and GSK-3 has been a matter of debate in the last few years, with some reports suggesting a positive link and others suggesting exactly the opposite scenario (i.e. GSK-3 activity as necessary for NF-B activation). In the only published study on cultured rat hepatocytes, Schwabe and Brenner (2002) analysed the experimental problem of TNF␣-induced apoptosis (a different approach to our present one). They described that inhibition of GSK-3 by LiCl resulted in an increased sensitivity to TNF␣-induced apoptosis,
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Fig. 10. Schematic representation of major signaling events connecting inhibition of GSK-3 and resulting -catenin nuclear translocation to increased transcription of TrCp1 that, in turn, leads to NF-B-dependent iNOS induction (see text for more details).
suggesting that GSK-3 inhibition was related to downregulation of NF-B dependent gene iNOS. However, this reported “indirect relationships” between GSK-3 and NF-B deserves some comments. First, in their experimental model the driving force for iNOS induction was TNF␣ itself, which is known to also activate NF-B. As a matter of fact, as authors clearly underlined, that study was reporting a down regulation of TNF␣-dependent event but, surprisingly, inhibition of GSK-3 by LiCl was not followed neither by inhibition of TNF␣-dependent NF-B DNA binding nor alteration in IB-␣ degradation, IB kinase activity or p65 nuclear import/export. Surprisingly, no changes in NF-B related parameters have been documented in the presence of LiCl alone; moreover, these results were puzzling since -catenin signaling activation through GSK-3 inhibition usually operates as a survival event (Calvisi, Ladu, Factor, & Thorgeirsson, 2004; Emanuele et al., 2004; Hahn et al., 2006; Sinha et al., 2005). Our data, however, are in agreement with a number of more direct literature findings obtained in different experimental models or cell lines. Wang, Adhikari, Li, Guan, and Hall (2004) have described that upregulation of -TrCp1 can accelerate the rate of IB-␣ degradation, leading to increased NF-B activity in vascular smooth muscle cells. Along these lines, Bournat, Brown,
and Soler (2000) described that activation of Wnt/catenin signaling pathway in PC12 cells resulted in NF-B activation, a condition that has been conceptually reproduced by others using LiCl to inhibit GSK-3 in human intestinal epithelial cells (Nemeth et al., 2002) or in renal medullary interstitial cells (Rao, Hao, & Breyer, 2004). Even more important, a direct in vivo relationship between -catenin and iNOS induction has been very recently reported (Du et al., 2006) in which hepatic levels of both iNOS and nuclear -catenin were found to be significantly increased after administration of LiCl. Similarly, in a non-hepatic “in vivo” experimental model, adherens junction disruption was again followed by the sequence of -catenin activation and increased expression of iNOS (Lee, Mruk, Wong, & Cheng, 2005). Acknowledgements Financial support was from Regione Piemonte and Fondazione CRT (Torino, MP), University of Torino (MP, SC) and MIUR (Rome, PRIN project, SC). References Barth, A. I., Nathke, I. S., & Nelson, W. J. (1997). Cadherins, catenins and APC protein: interplay between cytoskeletal complexes and signaling pathways. Curr. Opin. Cell. Biol., 9, 683–690.
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