INRS-Institut Armand-Frappier, Université du Québec, 245 Hymus Boulevard, Pointe-Claire, Québec, Canada, H9R 1G6
1 To whom correspondence should be addressed at INRS-Institut Armand Frappier, Université, 245 Hymus Boulevard Université du Québec, Pointe-Claire, Canada QC, H9R 1G6. Fax (514) 630-8850. E-mail: michel.charbonneau{at}iaf.inrs.ca, daniel.cyr{at}iaf.inrs.ca.
Received July 28, 2005; accepted September 9, 2005
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ABSTRACT |
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Key Words: Carcinogenesis; junction; E-cadherin; ß-catenin; glycogen synthetase kinase-3ß; connexins.
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INTRODUCTION |
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It has been proposed that there is a functional relationship between adherens and gap junctions. Adherens junctions are formed by cadherins, a family of single pass calcium-dependant transmembrane proteins. Cadherins interact in a homophilic manner with identical cadherins from adjacent cells. Cadherins are anchored to the cytoskelton via another family of proteins, the catenins. In certain malignant tumors, both adherens and gap junctions are downregulated, suggesting the existence of common pathways regulating the expression of these intercellular junctions (Fujimoto et al., 1997; Terzaghi-Howe et al., 1997
).
The integrin-linked kinase (ILK) pathway has been shown to be induced in many tumors. This pathway involves a phosphorylation signaling cascade leading to the regulation of genes involved in the control of cellular proliferation, differentiation, and cellcell interactions (Hannigan et al., 2005). The ILK is an evolutionarily conserved protein kinase, implicated in both integrin and growth-factor signaling. The basal activity of ILK is usually low, but it can be stimulated by cellular interactions and growth factors (Wu and Dedhar, 2001
). High levels of ILK have been observed in tumors of different tissues, notably the colon, ovary, prostate, and breast (Persad and Dedhar, 2003
). The activation of the ILK pathway can downregulate the expression of E-cadherin in intestinal and mammary epithelial cells (Novak et al., 1998
; Tan et al., 2001
). Because ILK can alter the expression of E-cadherin, and because E-cadherin has been linked to regulation of gap junctions in certain tissues, we hypothesized that HCB may be acting via the ILK pathway to regulate cellcell interactions in the liver.
Thus, the objectives of this study were to (1) determine whether the ILK pathway is activated by HCB in female rat liver, by assessing the expression and activity of the various components of this signaling pathway; (2) determine if E-cadherin levels are altered by HCB; and (3) using a cell line model, determine if the activation of the ILK pathway is directly linked to the inhibition of both E-cadherin and Cx32 in hepatocytes.
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MATERIALS AND METHODS |
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Rats were administered corn oil (vehicle control; n = 6) or HCB (100 mg/kg; n = 5) by gavage every day for 5 consecutive days, followed by a period of 45 days without treatment. Rats were sampled on day 50 of the experiment. This experimental model and time points were previously used to show that HCB caused a significant decrease in hepatic Cx26 and Cx32 in female rats prior to the induction of tumor formation at day 100 (Plante et al., 2002). Hexachlorobenzene administered to rats under this experimental protocol does not cause liver injury, as determined by the absence of morphological changes and normal plasma levels of alanine aminotransferase.
At the time of sampling, rats were anesthetized with isoflurane inhalation and the livers removed; a piece of the left lobe was embedded in Cryomatrix (Fisher Scientific, Mississauga, ON, Canada) for cryosections. Other lobes of the liver were frozen in liquid nitrogen and stored at 80°C for subsequent analysis.
Cell cultures.
MH1C1 rat liver cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2. In a first series of experiments, MH1C1 were exposed to either DMSO (0,1%, vehicle) or HCB (30 µM or 50 µM) for 7 days. These concentrations did not affect cell viability, as determined by trypan blue exclusion assays and methylthiazoletetrazolium (MTT) colorimetric analysis.
For experiments with GSK3ß inhibitors, cells at 95% confluence were kept for 16 h in DMEM without serum. Cells were then exposed to either DMSO or a noncytotoxic concentration of SB-415286 (125 nm or 150 nM) or kenpaullone (5 µM or 10 µM) for 24 h. Cells were then washed with ice-cold phosphate buffered saline (PBS), scraped, and lysed in buffer (25 mM HEPES pH 7.5; 150 mM NaCl; 10 mM MgCl2; 1 mM EDTA pH 7.0; 0.1% Triton X-100; 10% glycerol; 1 mM sodium orthovanadate). Lysates were sonicated twice and centrifuged, and the supernatant was collected. Aliquots were taken to assess protein content using the Bio-Rad Protein Assay reagents (Bio-Rad Laboratories, Mississauga, ON, Canada). Total cellular RNA was isolated using the phenol-chloroform method of Sambrook and Russell (2001).
Western blot analyses.
Frozen liver samples were homogenized in buffer (1:3 g/ml; 0.25 mM sucrose; 10 mM Tris (pH 7.5); 50 mg/ml leupeptin; 50 mg/ml aprotinin; 25 mg/ml pepstatin; 50 mg/ml antipain; 2.5g/ml phenylmethylsulfonyl fluoride (PMSF); 100 mM sodium orthovanadate) and centrifuged at 10,00 x g for 10 min at 4°C. The resulting supernatant was removed and its protein content determined using the Bio-Rad protein assay reagents.
Protein samples (75 mg for females and 125 µg for males) were diluted in Laemmli buffer (50 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate (SDS), 10% glycerol, 0.1% bromophenol blue, 100 mM 1,4-dithiothreitol (DTT)), and loaded onto either 8% or 12% polyacrylamide gels with a 4% stacking gel. Proteins were then separated by electrophoresis until the dye front reached the end of the gel, after which they were transferred onto a nitrocellulose membrane. Incubations were done either overnight at 4°C ( (ILK (1.02.0 mg/ml; Upstate, Chicago, IL); Akt (0.4 mg/ml; Santa Cruz Biotechnology, Santa Cruz, CA); phospho-Akt (2.5 mg/ml; Biosource International, Camarillo, CA); E-cadherin (0.8 mg/ml; Santa Cruz Biotechnology); Snail (2 mg/ml; Santa Cruz Biotechnology) or for 1 h at room temperature (GSK3ß [0.16 mg/ml; BD Transduction Laboratories, Lexington, KY]; phospho-GSK3ß [0.02 mg/ml; Cell Signaling Technology, Beverly, MA]). The membranes were washed in Tris (100 mM)-sodium chloride (154 mM) buffer (TBS) containing 0.05 % Tween and incubated for 1 h at room temperature with the appropriate secondary antibody (anti-rabbit conjugated to peroxidase [0.080.2 µg/ml; Santa Cruz Biotechnology]; anti-goat conjugated to peroxidase [0.3 µg/ml; Santa Cruz Biotechnology]; or anti-mouse-IgG conjugated to peroxidase [4.5 µg/ml; Sigma-Aldrich, Toronto, ON, Canada]). Signal detection was done by chemiluminescence using a commercial kit (Lumilight, Roche Diagnostic, Laval, QC, Canada).
Protein loading was standardized by measuring actin levels, using a murine monoclonal anti-actin antibody (1.0 µg/ml; Sigma-Aldrich). The membranes containing the proteins were washed and subsequently incubated for 1 h at room temperature with the secondary antibody (3.6 µg/ml; anti-mouse-IgG conjugated to peroxidase; Sigma-Aldrich). Signal detection was done by chemiluminescence using the Lumilight kit (Roche Diagnostic).
Flow cytometric analyses.
Intact nuclei were isolated according to the method of Sambrook and Russell (2001). Briefly, frozen liver samples were homogenized in buffer (10 mM HEPES (pH 7.6); 25 mM potassium chloride; 1 mM EDTA (pH 8.0); 2 M sucrose; 10% glycerol; 1 mM DTT; 0.5 mM PMSF; 1 µg/ml leupeptin; 1 µg/ml pepstatin; 0.15 mM spermine; 0.5 mM spermidine) and placed on a cushion of 10% sucrose in homogenization buffer. Samples were then centrifuged at 103,900 x g for 40 min at 4°C. Pellets containing the nuclei were resuspended in buffer (50 mM Tris (pH 8.3); 5 mM magnesium chloride; 0.1 mM EDTA (pH 8.0); 40% glycerol) and stored at 80°C.
Nuclei were fixed in absolute ethanol for 5 min at 20°C and incubated with blocking solution (137 mM NaCl, 3 mM KCl, 10 mM NaH2PO4, 2 mM potassium phosphate containing 5% bovine serum albumin (BSA), and 5% goat serum (pH 7.4)) for 15 min at 37°C. Nuclei were then incubated for 2 h at 37°C with a primary antibody (8 µg/ml goat anti-ß-catenin or 8 µg/ml goat anti-Akt; Santa Cruz Biotechnology) in blocking solution. The samples were washed three times with a saline solution (137 mM NaCl, 3 mM KCl, 10 mM NaH2PO4, 2 mM KH2PO4), incubated for 15 min at 37°C in blocking solution, and then incubated for 45 min at 37°C with a secondary antibody (4 µg/ml anti-goat-IgG conjugated to phycoerythrin, Sigma-Aldrich) in blocking solution. Nuclei were subsequently washed three times in the saline solution. Finally, nuclei were stained with 7-aminoactinomycin D for 15 min at 37°C and analyzed by flow cytometry (10,000 events in a Beckman Coulter system). Only positive events for 7-aminoactinomycin D staining were considered as nuclei (90% of all events in every sample) and used to compare either ß-catenin or Akt specific staining.
Immunohistochemical analyses.
Cryosections of liver were used to assess the localization of ß-catenin in control and HCB-treated female rats. Liver sections (6 µm) were fixed with methanol for 20 min at 20°C. The tissues were rehydrated in a saline solution for 20 min and blocked for 15 min at room temperature with blocking solution (saline solution containing 5% BSA and 5% goat-serum). Tissue sections were incubated with anti-ß-catenin antisera diluted in blocking solution (8 µg/ml, Santa Cruz Biotechnology) for 1.5 h at room temperature. Sections were once again washed twice in the saline solution containing Tween (0.05%), and then incubated with the secondary antibody (27.5 µg/ml anti-goat-IgG conjugated to fluorescein isothiocyanate conjugate, Jackson Laboratories, Bar Harbor, ME) for 45 min at room temperature in blocking solution. Tissues were washed three times in saline containing Tween (0.05%). Finally, nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and examined under a fluorescence microscope. The localization of ß-catenin was analyzed with the ImagePro Plus computer software (Media Cybernetics, Silver Spring, MD).
GSK3 activity assessment.
Frozen livers were homogenized in buffer (1:3 g/ml; 125 mM HEPES, pH. 7.4; 5 mM EDTA; 5 mM ethylene glycol-bis[2-aminoethylether]-N,N,N'N',-tetraacetic acid [EGTA]; 5 mM benzamidine; 1 mM PMSF; 1 mg/µl soybean tryspin inhibitor; 1 mM sodium orthovanadate; 1 mM sodium pyrophosphate; 0.05% sodium deoxycholate; 1% Triton; 0.1% ß-mercaptoethanol) and centrifuged at 14,000 x g for 10 min at 4°C. Protein content of the resulting supernatant was determined using the Bio-Rad Protein Assay reagents. An aliquot containing 360 µg of the sample was immunoprecipitated overnight at 4°C using an anti-GSK3ß antibody (16 µg/ml, BD Transduction Laboratories) bound to protein G Sepharose beads (Amersham Biosciences, Baie d'Urfée, QC, Canada). Resulting immunocomplexes (25 µl) were then incubated for 45 min in 50 µl of GSK3ß-assay buffer (50 mM Tris (pH. 7.4); 1 mM EGTA; 0.15 mM NaCl; 0.03% Brij-35; 0.1% ß-mercaptoethanol; 50 mM MgCl2) containing 6 mmol of phospho-glycogen synthase peptide-2 (PEP2) or glycogen synthase peptide-2 (Ala21) (Upstate) and 10 mCi/ml [gamma-32P] ATP (PerkinElmer, QC, Canada) for 45 min at 37°C. The reaction was stopped by the addition of 10 µl of tricine sample buffer (200 mM tricine; 2% SDS; 40% glycerol; 0.08% Coomassie brilliant blue).
Samples were loaded onto a 15% tricine gel with a 5% stacking gel. Electrophoresis was performed at 120 V until the dye front reached the end of the gel. The gel was then placed into an autoradiographic cassette. The resulting autoradiograms were analyzed using a densitometer (Fluor-S MultiImager, Bio-Rad Laboratories).
Reverse transcription-polymerase chain reaction.
Total cellular RNA was isolated from liver by the guanidinium isothiocyanate method of Chomczynski and Sacchi (1987) and from MH1C1 cells by the phenol-chloroform extraction method of Sambrook and Russell (2001)
. The RNA was reverse transcribed using an oligo d(T)1618 primer (Amersham Pharmacia Biotech) and M-MLV reverse transcriptase (Canadian Life Technologies) according to the manufacturers' instructions. The cDNA templates (500 ng) were amplified for Cx32 (reverse primer: CAG GCT GAG CAT CGG TCG CTC TT; forward primer: CTG CTC TAC CCG GGC TAT GC) using 30 cycles of a two-step PCR; denaturation at 94°C for 30 s, annealing and elongation at 69°C for 60 s. E-cadherin was amplified with specific primers (reverse primer: CGG TTG CCC CAT TCG TTC AGA; forward primer: TGC CCC AGT ATC GTC CCC GTC) using 35 cycles of denaturation at 94°C for 30 s, annealing at 69°C for 30 s, and elongation at 72°C for 60 s. Snail was amplified with specific primers (reverse primer: GCC CAG GCT GAG GTA CTC C; forward primer: CTT CCA GCA GCC CTA CGA CCA) using up to 45 cycles of denaturation at 94°C for 30 s, annealing at 61.6°C for 30 s, and elongation at 72°C for 30 s. The RT-PCR products were separated on either a 1% or 2% agarose gel and visualized by ethidium bromide staining. Cx32 and E-cadherin signals were standardized by GAPDH amplification (reverse primer: GCC GGG ACA GGC GGC AGG TTA G; forward primer: GGG TGA GGT GAG CAT GGA GGA CG).
Statistics.
Statistical differences between control and HCB-treated groups were determined with Student's t-test. Statistical differences between groups in in vitro experiments were determined by analysis of variance (ANOVA), followed a posteriori by a Tukey test for multiple comparisons between experimental groups. Each experiment was repeated three times using three or four independent samples per group. Significance was set at p < 0.05. All statistical analyses were done using the SigmaStat computer software (Jandel Scientific Software, San Rafael, CA).
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RESULTS |
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DISCUSSION |
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Activation of the ILK pathway in tumors has been observed in various types of tissues, but until now it had not been reported for the liver (Hannigan et al., 2005). Moreover, this is the first report of chemically induced activation of the ILK pathway. Indeed, our results show that in the liver of female rats treated with HCB, there is an increased level of active ILK.
Integrins are dimeric cell adhesion receptors present at the base of the plasma membrane that are implicated in the adhesion of the cell to the extracellular matrix and are involved in signal transduction (Dedhar, 2000). Stimulation of integrins by growth factors, such as EGF, increase ILK kinase activity. Interestingly, HCB has been reported to increase EGFR-tyrosine kinase activity in female rat livers (Randi et al., 2003
). High levels of ILK have been observed in various human tumors (Hannigan et al., 2005
). Intestinal and mammary epithelial cells overexpressing ILK adopt a highly invasive phenotype, which leads to tumor formation (Troussard et al., 2000
). Moreover, specific inhibitors of ILK attenuate the growth of human colon carcinoma cell lines (Hill and Hemmings, 2002
). Recent studies report that the intensity of ILK-specific immunostaining in ovarian tumors is positively correlated with the grade of the tumor (Ahmed et al., 2003
). In human colon cancers, where ILK signaling is dysregulated early in tumor development, it is suggested that ILK activation is an early event in carcinogenesis (Marotta et al., 2003
). In our HCB model, ILK expression was undetectable in the liver of control rats, but levels were significantly increased in these HCB-treated females. As such, changes in the ILK pathway are induced at a precancerous stage (non-initiated females), because tumor formation occurs only 1 year after the administration of DEN in HCB-treated female rats. This supports our earlier observations that in pre-cancerous HCB-treated females, the liver undergoes a number of structural and physiological changes predisposing it to tumor formation (Plante et al., 2002
). Furthermore, while we observed a significant increase in the expression of ILK in HCB-treated female rats, there were no differences between control and treated males, suggesting that the effects we observed are directly related to the gender-specific tumor development.
Akt is a downstream effector of ILK. Akt overexpression has been reported in ovarian, breast, prostate, and pancreatic cancers. Once activated, Akt is first targeted to the plasma membrane, where it can be either phosphorylated by itself or by other enzymes. The hyperphosphorylated form of Akt is then translocated into the nucleus, where it can initiate transcription (Saji et al., 2005). In the present study, the increased phosphorylation of Akt appears to be related to its nuclear translocation in the liver of treated animals, as it is associated with the nuclear fraction of cells in HCB-treated females (Figs. 2 and 3). Nuclear Akt can act either directly as a transcription factor or as a kinase to phosphorylate other transcription factors (Andjelkovic et al., 1997
; Meier et al., 1997
; Nicholson and Anderson, 2002
; Saji et al., 2005
;). Nuclear Akt results in the activation of cell survivor factors that prevent apoptosis, as well as in the expression of cell cycle progression genes (e.g., Bcl-2, Bcl-x and IKK, caspase 9, and members of the BH3 (Bad, Bid, Bik), Bax (Bax and Bak), and Forkhead families) (Saji et al., 2005
; Strange et al., 2001
).
GSK3ß has been implicated in a number of intracellular signaling pathways, including both the ILK and the WNT pathways (Nusse, 2005). GSK3ß can be phosphorylated on serine 9 by either Akt or ILK, resulting in its inactivation (Dedhar, 2000
). In mammary tumors in which ILK is overexpressed, there is a dramatic increase in serine 9 phosphorylation of GSK3ß and thus its inactivation (Marotta et al., 2003
). The inactivation of GSK3ß has been linked to carcinogenesis through its regulation of apoptosis and cell cycle progression; overexpression of an inactive mutant of GSK3ß prevents apoptosis in pheochromocytoma cells and rat-1 fibroblasts (Frame and Cohen, 2001
). In contrast, the activation of GSK3ß is known to phopshorylate cyclin D1, thereby targeting it for degradation by the ubiquitinproteosome complex, resulting in an inhibition of the cell cycle (Diehl et al., 1998
). In the present study, although there were no changes in total GSK3ß protein levels, the levels of the inactive phosphorylated form of the protein in HCB-treated female rats were more than two times higher than in controls. Likewise GSK3ß activity was also decreased in HCB-treated females rats. Inactivation of GSK3ß has been shown to occur in carcinogenesis, resulting in an alteration of ß-catenin localization (Frame and Cohen, 2001
). When GSK3ß is active cytosolic ß-catenin is ubiquinated, whereas when it is inactive, ß-catenin accumulates in the cytoplasm and translocates to the nucleus, where it can act as a transcriptional co-factor.
The implication of ß-catenin signaling has been reported for many different types of tumors and immortalized cell lines (Brabletz et al., 2002). The role of ß-catenin in these processes is multifactorial and involves either signaling pathways linked with its role as a cadherin-binding protein within the adherens junctional complex or as a cytosolic signaling molecule that can translocate into the nucleus and modulate specific gene transcription. The levels of ß-catenin are higher in tumors than in healthy cells that surround the tumors (Brabletz et al., 2002
; Morin, 1999
). Mutations in the ß-catenin gene have been observed in both early and late stage tumors (Devereux et al., 1999
). ß-Catenin mutations in the N-terminal GSK3ß phosphorylation sites result in the absence of ß-catenin degradation, resulting in the accumulation of high levels of ß-catenin into the cytoplasm and its transfer to the nucleus, where it can act as a co-factor of the TEF/Lef transcription factor to modulate the expression of genes, including oncogenes implicated in cell proliferation (Gotoh et al., 2003
; Novak et al., 1998
). ß-Catenin levels can also be increased by the inactivation of GSK3ß (Morin, 1999
). Our results indicate that nuclear ß-catenin is increased in the liver of female rats treated with HCB. Immunohistochemical analyses clearly indicate that nuclear ß-catenin was not localized to a particular area of the liver but appeared to represent a more generalized effect. To assess the levels of nuclear ß-catenin, we developed a flow cytometric method that allowed us to quantify the levels of nuclear ß-catenin. These results indicate that there were almost twice as many ß-cateninpositive nuclei in HCB-treated female rat as compared to controls. Several studies have reported that increased nuclear ß-catenin is associated with a decrease in cell adhesion and a decrease in the expression of E-cadherin (Huber et al., 1996
). Recent studies suggested that the decrease of E-cadherin levels may not be related to ß-catenin nuclear translocation, but rather to the induction of Snail by GSK3ß and its subsequent effect on the regulation of E-cadherin transcription (Zhou et al., 2004
; Bachelder et al., 2005
).
Snail is a transcription factor containing a zinc finger motif that has been shown to inhibit E-cadherin transcription via its interaction with specific E-box response elements on the E-cadherin promoter (Peinado et al., 2004). Inhibition of GSK3ß by siRNA or with specific inhibitors stimulates Snail transcription and decreases E-cadherin levels in normal breast and skin epithelial cells (Bachelder et al., 2005
). Zhou et al. (2004)
have reported that there are two GSK3ß consensus phosphorylation motifs on Snail that determine its localization and degradation. In our present model, Snail was undetectable at both protein and mRNA levels in control and treated groups. Therefore the data do not support a major role for Snail in E-cadherin downregulation in HCB-treated female rats.
Decreased expression of E-cadherin by diverse mechanisms, including both genetic and epigenetic events, plays a significant role in multistage carcinogenesis In HCB-treated rats we observed a significant decrease in E-cadherin levels in females. Such a decrease in E-cadherin levels is often observed in cancer cells and has been associated with the capacity of tumors to grow and metastasize (Beavon, 2000). Loss of E-cadherin expression has been observed for a large proportion of tumors in the liver, as well as for several other organs (Beavon, 2000
). Decreases in E-cadherin expression are involved not only in late stages of human cancers but also in early phases of carcinogenesis. Thus, the downregulation of E-cadherin observed in HCB-treated rats is an important mechanistic event, as it may render the liver more susceptible to tumor progression. It has been proposed that this is a mechanism by which the progression of initiated cancerous cells (e.g., induced by DEN) to fully developed tumors occurs. Previous studies have suggested that the downregulation of E-cadherin may result from the phosphorylation of various effectors, including those regulated by integrin signaling pathways (Oloumi et al., 2004
). In the present study, the translocation of both Akt and ß-catenin into the nucleus and the reduction of E-cadherin suggest that either or both of these transcription factors may modify E-cadherin expression. A role for Akt in this process is supported by a recent study demonstrating that in squamous carcinoma cell lines engineered to constitutively express active Akt, levels of E-cadherin mRNA were significantly decreased (Grille et al., 2003
).
We have previously shown that in HCB-treated female rats there is a decrease in hepatic connexins at both the mRNA and protein levels (Plante et al., 2002). Therefore it is likely that a common pathway for the regulation of hepatic connexins and E-cadherin exists. Previous findings have indicated that adherens regulation and gap junction regulation are related, as both are often reduced or absent in many tumors (Fujimoto et al., 1997
; Terzaghi-Howe et al., 1997
).
To understand the mechanism by which HCB regulates both components of adherens and gap junctions in the liver, a rat hepatocarcinoma cell line, MH1C1, was used. This cell line expresses both E-cadherin and Cx32. Cells treated with HCB for 7 days displayed lower levels of both Cx32 and GSK3ß, suggesting that this model is appropriate to further study the mechanistic links between the ILK pathway and these cellular junctions. In our first series of experiments we used specific inhibitors of GSK3ß in MH1C1 cells. As expected, both SB-415286 and kepaullone inhibited GSK3ß activity. Inhibition of GSK3ß resulted in decreased levels of E-cadherin, but it had no effects on Cx32 levels. This suggests that while both types of cellular junctions are decreased by HCB, and while HCB results in decreased GSK3ß activity, the signaling cascade implicated in the inhibition of both E-cadherin and Cx32 is different. Because in vivo ILK activity is increased by HCB treatment, and because the effects on Cx32 appear to be independent of GSK3ß as shown in vitro, we postulate that Cx32 regulation by HCB occurs either downstream of ILK via the activation of a second sub-pathway or through another unidentified signaling pathway parallel to ILK. Also, because Akt is also activated in female rat livers in vivo, it may represent a potential signaling messenger by which Cx32 is regulated. Further studies will be needed to elucidate this possibility.
In conclusion, results from these experiments indicate a decrease in hepatic E-cadherin expression and an activation of the ILK signaling pathway in HCB-induced livers prior to the appearance of hepatic tumors, suggesting that these may represent early events in chemically induced hepatic carcinogenesis. Overall, by looking at changes in protein expression and phosphorylation status, we propose a mechanism by which HCB renders female rats more susceptible to tumor development by activating a pathway for cellcell interaction dysregulation by which both E-cadherin and connexins are modulated. This mechanism represents a novel pathway that may be activated by epigenetic carcinogens in order to promote tumor formation by disrupting cellcell interactions.
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ACKNOWLEDGMENTS |
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