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Carcinogenesis Advance Access originally published online on May 5, 2006
Carcinogenesis 2006 27(9):1923-1929; doi:10.1093/carcin/bgl059
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Activation of the integrin-linked kinase pathway downregulates hepatic connexin32 via nuclear Akt

Isabelle Plante, Michel Charbonneau and Daniel G. Cyr*

INRS-Institut Armand-Frappier, Université du Québec 245 Hymus Boulevard, Pointe-Claire, QC, Canada H9R 1G6

*To whom correspondence should be addressed. Tel: +1 514 630 8833/8831; Fax: +1 514 630 8850; Email: daniel.cyr{at}iaf.inrs.ca


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gap junctions mediate intercellular communication through channels composed of proteins termed connexins (Cxs). We have shown that Cx32 is downregulated in the liver of female rats exposed to hexachlorobenzene (HCB), an epigenetic environmental carcinogen. This is concomitant with the activation of the integrin-linked kinase (ILK) pathway, leading to the activation and nuclear translocation of Akt and the inactivation of glycogen synthase kinase-3ß (GSK3ß). E-cadherin, an adhering junction protein, is also downregulated in the liver of these female rats, owing to the inactivation of GSK3ß. Using an in vitro model, the aim of this study was to determine the role of the ILK pathway in the regulation of Cx32. In order to mimic the activation of the ILK pathway, a well-differentiated rat hepatoma cell line, MH1C1, was transiently transfected with an expression vector for ILK (ILK+ cells). ILK+ cells displayed significantly lower Cx32 mRNA levels and Akt was also activated and translocated into the nucleus. Using a constitutively active Akt expression vector, we showed that Akt transfected cells had lower Cx32 mRNA levels, indicating a role for Akt in Cx32 regulation. Finally, using an Akt-NES vector, a nuclear-active form of Akt, we showed that Cx32 protein levels were reduced in transfected cells as compared with cell transfected with the wild-type inactive Akt vector, suggesting that the nuclear form of Akt is responsible for the downregulation of Cx32. Overall, these data indicate that Cx32 is downregulated by the ILK pathway activation in rat hepatocytes and that this is mediated via the activation and nuclear translocation of Akt.

Abbreviations: Akt-NES–GFP, green-fluorescent protein containing a mutated nuclear only active form of Akt; Akt-WT–GFP, green-fluorescent protein containing a wild-type inactive Akt; Cxs, connexins; EGFR, epidermal growth factor receptor; GJIC, gap junctional intercellular communication; GSK3ß, glycogen synthase kinase-3ß; HCB, hexachlorobenzene; ILK, integrin-linked kinase; PBS, phosphate-buffered saline; RT–PCR, reverse transcription–polymerase chain reaction


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
It has been reported that in several types of cancers a decrease in gap junctional intercellular communication (GJIC) is associated with the development of tumors. Gap junctions are intercellular channels formed by proteins termed connexins (Cxs). Gap junctions allow direct communication between cells by allowing the exchange of small molecules (≤1 kDa) between neighboring cells (1). It has been shown that epigenetic carcinogens can promote tumor formation by downregulating Cxs and consequently GJIC (2). However, the intracellular signaling pathways implicated in the regulation of Cxs are poorly understood.

Adhering junctions are implicated in cell–cell interactions and in intracellular signalization. They are formed primarily by a large family of single-pass calcium-dependent transmembrane proteins termed cadherins (3). It has been suggested that there exists a functional relationship between adhering and gap junctions and, furthermore, that in certain tissues adhering junctions are necessary to allow binding of adjacent cellular plasma membranes. This subsequently allows the Cxs from each cell to bind and form the intercellular pores of the gap junction. In certain cancers, both adhering and gap junctions are downregulated, suggesting the existence of common pathways controlling the expression of these two types of intercellular junctions (4,5).

The activation of the integrin-linked kinase (ILK) pathway has been shown to downregulate the expression of E-cadherin in intestinal and mammary epithelial cells as well as in human colon carcinoma cell lines (6). The ILK pathway is a phosphorylation signaling cascade leading to the regulation of genes involved in the control of cellular proliferation, differentiation and cell–cell interactions (7). The ILK phosphorylation cascade can modulate changes in gene expression via the activation of nuclear transcription factors (6,7).

We have demonstrated that exposure to hexachlorobenzene (HCB), an epigenetic environmental carcinogen, resulted in significantly lower levels of both Cx32 and Cx26 as well as GJIC in the livers of female rats sampled 45 days after the administration of HCB (8). However, Cxs and GJIC in males were unaltered. These changes coincided with a downregulation of E-cadherin induced by the activation of the ILK signaling pathway (5). Using both in vivo and in vitro approaches we were able to demonstrate that the ILK pathway was activated in the liver of HCB-treated rat females and that this activation decreased the expression of E-cadherin via inactivation of glycogen synthase kinase-3ß (GSK3ß), a downstream target of ILK. However, Cx32 levels were not affected by the inactivation of GSK3ß in vitro, although Cx32 mRNA levels were decreased by 50% in HCB-treated cells as compared with the untreated control cells. These results suggested that the regulation of Cx32 by HCB occurs either downstream of ILK via a second pathway or through another unidentified signaling pathway parallel to ILK (5). In HCB-treated female rats, hepatic nuclear Akt protein levels were increased. It has been shown that once activated by phosphorylation, including ILK-induced phosphorylation, Akt phosphorylates different proteins into the cytoplasm and can then translocate into the nucleus where it phosphorylates transcription factors and modulates gene expression (9,10).

Since HCB exposure downregulates hepatic Cx expression in rat both in vivo and in vitro, and since E-cadherin is decreased by HCB-induced ILK pathway activation, it is possible that Cx levels are also modulated by the same pathways, but using an intermediate other than GSK3ß. Consequently, the objective of this study was to assess whether or not the activation of the ILK pathway is implicated in hepatic Cx32 regulation.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture conditions
Rat liver cells (MH1C1) 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.

Transfection and vectors
An ILK expression vector pcDNA3.1/V5-his A was generously provided by Dr S. Dedhar (University of British Columbia). A constitutively active myr-Akt1 expression vector pUSEamp(+) was purchased from UpState Biosciences (Chicago, IL). The Akt-NES–GFP (green-fluorescent protein containing a mutated nuclear only active form of Akt) and Akt-WT–GFP (green-fluorescent protein containing a wild-type inactive Akt) expression vectors pEGFP-C1 were generous gifts from Dr M.D. Ringel (Ohio State University). Cells (95% confluence) were transiently transfected using 40 µg/ml lipofectamine 2000 (Invitrogen Life Technologies) in DMEM without FBS, according to the manufacturer's instructions. Cells were lysed 24 h later, and total protein or total RNA was isolated from the lysate. PCL-neo (Promega, Madison, WI) was used as a control vector, and is represented as control group on the graphs. Table I summarizes the different vectors used in this study.


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  		Table I List of transfection vectors used in Akt experiments and their activity

 
Western blot analysis
Cytoplasm and nuclear/membrane proteins were extracted from MH1C1 cells using the NE-PER Cytoplasmic and Nuclear Protein extraction kit (Pierce Biotechnology, Rockford, IL, USA). The purity of the nuclear enrichment fraction was determined using albumin as a marker for the cytoplasmic fraction and the transcription factor Sp1 as a marker for the nuclear fraction (Figure 1). Overexposed films show <6% cross-contamination between cytoplasmic and nuclear extracts (data not shown). Protein levels were determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Mississauga, ON, Canada). A 50 µg aliquot of total proteins was subjected to SDS–polyacrylamide gel electrophoresis (PAGE) on either a 10 or 12% gel and subsequently transferred onto a nitrocellulose membrane. Membranes containing the transferred proteins were blocked with phosphate-buffered saline (PBS) containing 5% powdered milk and 0.05% Tween and then hybridized overnight at 4°C with the appropriate primary antibody (rabbit anti-ILK, 1.0–2.0 mg/ml, Upstate, Chicago, IL; goat anti-Akt, 0.4 mg/ml, Santa Cruz Biotechnology, Santa Cruz, CA; rabbit anti-phospho-Ser473–Akt, 2.5 mg/ml, Biosource International, Camarillo, CA; rabbit anti-Sp1, 0.4 mg/ml, Santa Cruz Biotechnology, Santa Cruz, CA; and rabbit anti-albumin, 2.8 mg/ml, DakoCytomation, Denmark). Following the hybridization, the membranes were washed in PBS containing 0.05% Tween and incubated for 1 h at room temperature with the appropriate peroxidase-conjugated secondary antibody (anti-rabbit, 0.08–0.2 µg/ml, Santa Cruz Biotechnology; anti-goat, 0.3 µg/ml, Santa Cruz Biotechnology; anti-mouse-IgG, 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).


Figure 1
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  		Fig. 1 Determination of cross-contamination between nuclear and cytoplasmic fractions. Cytoplasm (C) and nuclear (N) proteins were extracted from MH1C1 cells using the NE-PER Cytoplasmic and Nuclear Protein extraction kit (Pierce Biotechnology, Rockford, IL, USA). An aliquot of protein (50 µg) from each sample was subjected to western blot analysis. Nuclear compartment enrichment has been determined using albumin as a cytoplasmic marker and Sp1 transcription factor as a nuclear marker.

 
Cytoplasmic 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). Nuclear protein loading was verified using Ponceau red (5% Ponceau red in 5% acetic acid) coloration and quantified using Fluor-S MultiImager Bio-Scan transilluminator (Bio-Rad Laboratories).

Immunoprecipitation
Aliquots (50 µl) of protein G-Agarose (Roche Diagnostic) were incubated overnight at 4°C with 8 µg of murine anti-Cx32 antibody (Sigma-Aldrich). Total proteins (100 µg) for each sample were added and incubated for an additional hour at 4°C. Agarose beads were collected by centrifugation and washed in PBS. The agarose beads were then incubated in Laemmli sample buffer and boiled for 10 min. The resulting proteins were then subjected to SDS–PAGE on a 12% gel and subsequently transferred onto a nitrocellulose membrane. Membranes containing the transferred proteins were blocked with PBS containing 5% powdered milk and 0.05% Tween and then hybridized overnight at 4°C with anti-mouse Cx32 (Sigma-Aldrich). Following the hybridization, the membranes were washed in PBS containing 0.05% Tween and incubated for 1 h at room temperature with peroxidase-conjugated anti-mouse-IgG (4.5 µg/ml; Sigma-Aldrich). Signal detection was done by chemiluminescence using a commercial kit (Lumilight, Roche Diagnostic).

Reverse transcription–polymerase chain reaction (RT–PCR)
Total cellular RNA was isolated from MH1C1 cells using the phenol–chloroform extraction method of Sambrook and Russell (11). The RNA was reverse-transcribed using an oligo d(T)16–18 primer (Amersham Pharmacia Biotech) and MMLV reverse transcriptase (Canadian Life Technologies) according to the manufacturer's 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, and annealing and elongation at 69°C for 60 s. ILK was amplified with specific primers (reverse primer: TTG AGC TTT GCC AGG AAG TT; forward primer: ATG TGA TGA ATC GTG GGG AT). RT–PCR products were separated on either a 1 or 2% agarose gel and visualized by ethidium bromide staining. Cx32 mRNA levels were standardized using GAPDH expression as a standard (reverse primer: GCC GGG ACA GGC GGC AGG TTA G; forward primer: GGG TGA GGT GAG CAT GGA GGA CG).

Immunofluorescence microscopy
MH1C1 cells transfected with the Akt-NES–GFP and Akt-WT–GFP expression vector were fixed 10 min with cold methanol at –20°C and permeabilized with PBS–Triton (0.3%) for 15 min at room temperature. Nuclei were stained using 6-diamidino-2-phenylindole (DAPI) and examined under a fluorescent microscope. The localization of Akt was visualized using the ImagePro Plus computer software (Media Cybernetics, Silver Spring, MD).

Flow cytometric analyses
MH1C1 cells were transfected with the Akt-NES–GFP and Akt-WT–GFP expression vectors. A day (24 h) later the cells were fixed for 10 min in cold methanol at –20°C and then permeabilized with PBS–Triton (0.3%) for 15 min at room temperature. Cells were blocked using PBS–BSA (bovine serum albumin) (5%) for 30 min at 37°C and then incubated for 90 min at 37°C with a mouse anti-Cx32 antibody (Sigma-Aldrich) diluted in PBS–BSA (5%). The cells were then washed with PBS and incubated for 45 min at room temperature with goat anti-mouse-phycoerythrin labeled antibody (Jackson Laboratories, Bar Harbor, MA). Cx32 expression was quantified using flow cytometry (50 000 events). Only positive events in GFP stained cells were used to analyze Cx32 protein levels using a FACSCalibur system.

Statistics
Statistical differences between groups were determined by ANOVA followed a posteriori by a Turkey test for multiple comparisons between experimental groups. Each experiment was done in triplicate using 3–4 independent samples per group (n = 12). Significance was established at P < 0.05. All statistical analyses were done using the SigmaStat computer software (Jandel Scientific Software, San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
In order to determine if HCB-induced downregulation of Cx32 observed in vivo results from the activation of the ILK pathway, MH1C1, a well-differentiated rat hepatoma cell line, was transiently transfected with an ILK expression vector in order to overexpress ILK. Using RT–PCR, we were able to confirm that the expression of ILK was increased in cells transfected with the ILK expression vector. In these cells (MH1C1–ILK+), ILK mRNA levels were 2-fold higher than in cells transfected with the negative control vector (Figure 2A), whereas ILK protein levels were increased 3-fold, as determined by western blot analysis (Figure 2B).


Figure 2
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  		Fig. 2 ILK mRNA and protein levels in MH1C1 and MH1C1–ILK+ cells. MH1C1 cells were transiently transfected with either control or ILK expression vectors and lysed 24 h later. (A) mRNA was extracted and used for semi-quantitative RT–PCR analysis using ILK-specific primers. Samples were standardized for loading using GAPDH. Data are expressed as the mean ± SEM (n = 12, from three independent experiments). **P < 0.01 indicates a significant difference from controls. (B) Cells were lysed, and an aliquot of protein from each sample was subjected to western blot analysis using ILK antisera. Amounts of protein loaded were 50 µg. Data were standardized for loading using an actin antisera. Data are expressed as the mean ± SEM (n = 12, from three independent experiments). *P < 0.05 indicates a significant difference from controls.

 
Our previous studies have shown that HCB can stimulate ILK in rat liver and that this is associated with decreased Cx32 levels. In this study, we observed a 2-fold decrease in Cx32 mRNA levels in MH1C1–ILK+ cells relative to controls (Figure 3). This indicates that the activation of the ILK pathway results in a downregulation of Cx32 in rat hepatocytes.


Figure 3
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  		Fig. 3 Connexin32 mRNA levels in MH1C1 and MH1C1–ILK+ cells. MH1C1 cells were transiently transfected with either control or ILK expression vectors and lysed 24 h later. mRNA was extracted using the phenol–chloroform method and then used for RT–PCR analysis using Cx32 specific primers. Data were standardized for loading using GAPDH mRNA levels. Data are expressed as the mean ± SEM (n = 12, from three independent experiments). **P < 0.01 indicates a significant difference from controls.

 
Our previous results showed that Akt, a downstream target of ILK, was increased in hepatic nuclei of HCB-treated females, suggesting its activation by ILK. Akt can be activated by phosphorylation at the plasma membrane by a variety of kinases, including ILK. Western blot analysis indicated that whereas there was no change in cytoplasmic Akt protein levels in MH1C1–ILK+ (Figure 4A), there was a ~4-fold increase in nuclear/membrane Akt protein levels as compared with untransfected cells (Figure 4B). Using a phospho-specific anti-Akt antibody, we then showed that there was also a ~2-fold increase in phospho-Akt cytoplasmic protein levels in MH1C1–ILK+ cells as compared with the control (Figure 4C). Furthermore, there was a 4-fold increase of phospho-Akt nuclear/membrane protein levels in MH1C1–ILK+ cells as compared with the control (Figure 4D). These results indicate that the overexpression of ILK and subsequent activation of the ILK pathway in rat hepatocytes can activate the Akt intracellular pathway via an increase in Akt phosphorylation. As such, this provides a functional experimental tool to assess the role of ILK and Akt on gap junctional proteins. The ILK pathway can either activate Akt, as described above, or inactivate GSK3ß and promote the nuclear translocation of cytoplasmic ß-catenin. Our previous work has also demonstrated that chemical inhibitors of GSK3ß failed to alter Cx32 levels in MH1C1 cells, suggesting that ILK is acting via another cellular target, such as Akt.


Figure 4
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  		Fig. 4 Akt and phospho-Akt protein levels in MH1C1 and MH1C1–ILK+ cells. MH1C1 cells were transiently transfected with either control or ILK expression vectors and lysed 24 h later. Cells were lysed using the NE-PER Cytoplasmic and Nuclear Protein extraction kit (Pierce Biotechnology), and an aliquot of protein (50 µg) from each sample was subjected to western blot analysis using (A and B) Akt antisera or (C and D) phospho-Akt antisera. Data were standardized for loading using an actin antisera for cytoplasmic fractions. Data are expressed as the mean ± SEM (n = 12, from three independent experiments). *P < 0.05, **P < 0.01 and ***P < 0.005 indicate a significant difference from controls.

 
In order to determine if hepatic Cx32 downregulation occurred via the activation of Akt by ILK, we mimicked ILK-induced Akt activation by transfecting MH1C1 cells with an expression vector containing an active mutated form of Akt (MH1C1–Akt+). This vector expresses a constitutively active form of Akt owing to the presence of 11 N-terminal amino acid residues of avian c-src that are required for protein myristoylation at the amino terminus of Akt. This signal sequence allows Akt to be translocated to the plasma membrane where it can be activated by phosphorylation. We first determined the efficiency of the transfection using western blot analysis. In all samples, a band at 60 kDa, corresponding to endogenous Akt, was observed (Figure 5). A second heavier band was detected (~64 kDa) in the fractions of MH1C1–Akt+ cells, corresponding to the exogenous mutated form of Akt produced by the vector. A marked increase in exogenous Akt was present in the cytoplasmic and membrane fraction of MH1C1 cells transfected with the Akt vector (MH1C1–Akt+) (Figure 5). In these cells, however, there was also an increase in Akt protein levels in the nuclear/membrane fraction of MH1C1–Akt+, suggesting the activation and increased expression of Akt (Figure 5). There was also a substantial increase in active phospho-Akt in both the cytoplasmic and nuclear/membrane fractions of MH1C1–Akt+ cells as compared with MH1C1 cells, confirming the transfection of the phosphorylated active form of Akt (Figure 5). To determine the role of Akt in Cx32 regulation, we then measured Cx32 mRNA levels in MH1C1 and MH1C1–Akt+ cells. Using semi-quantitative RT–PCR, we observed a 40% decrease in Cx32 mRNA levels (Figure 6). Cx32 protein levels were determined by western blot analyses following Cx32 immunoprecipitation. Results indicate a 40% decrease in Cx32 protein levels in MH1C1–Akt+ cells as compared with MH1C1 cells. These results suggest that the activation of Akt results in the downregulation of Cx32 expression at both the mRNA and protein levels.


Figure 5
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  		Fig. 5 Akt and phospho-Akt protein levels in MH1C1 and MH1C1–Akt+ cells. MH1C1 cells were transiently transfected with either control or active Akt expression vectors and lysed 24 h later. Cells were lysed using NE-PER Cytoplasmic and Nuclear Protein extraction kit (Pierce Biotechnology), and an aliquot of protein (50 µg) from each sample was subjected to western blot analysis using Akt antisera or phospho-Akt antisera. Data were standardized for loading using an actin antisera for the cytoplasmic fractions.

 

Figure 6
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  		Fig. 6 Connexin32 mRNA and proteins levels in MH1C1 and MH1C1–Akt+ cells. MH1C1 cells were transfected with either control or active Akt expression vectors and lysed 24 h later. (A) mRNA was extracted using the phenol–chloroform method and then used in RT–PCR analysis using Cx32 specific primers. Data were standardized for loading using GAPDH mRNA levels. Data are expressed as the mean ± SEM (n = 12, from three independent experiments). (B) Proteins were extracted and Cx32 immunoprecipitated (IP) as described in Materials and methods. The nitrocellulose membrane containing IP Cx32 was subjected to immunoblotting using an anti-Cx32 antisera. Data are expressed as the mean ± SEM (n = 7) The experiment was repeated once. **P < 0.01 indicates a significant difference from controls.

 
Akt can act either at the membrane and cytoplasmic levels, where it can phosphorylate effector proteins, or at the nuclear level, where it can act as a transcription factor or phosphorylate transcription factor(s) (10). In order to determine if the decrease in Cx32 induced by Akt was due to nuclear translocation of Akt, we tested two other vectors coupled to a GFP containing either a wild-type inactive Akt (Akt-WT+) or a mutated nuclear only active form of Akt (Akt-NES+), in which the nuclear-export sequence was modified in order to retain Akt in the nucleus. Using immunofluorescent microscopy, we first demonstrated that in MH1C1–Akt-NES+ cells Akt was present in the nuclei of transfected cells, whereas in MH1C1–Akt-WT+ the GFP was distributed throughout the cytoplasm (Figure 7A). As a result of the low transfection rate observed with the Akt-NES+ vector (~5% of cells), we used flow cytometric approach to analyze the expression of Cx32 only in those cells that expressed the nuclear Akt (Figures 7Bi and 7Bii). Whereas there were no differences in Cx32 levels between untransfected control cells and cells transfected with the WT inactive vector (MH1C1–Akt-WT+ cells) (Figure 7Biii), there was a significant 25% decrease in Cx32 protein levels in cells transfected with the nuclear only active vector (MH1C1–Akt-NES+) as compared with either MH1C1–Akt-WT+ or untransfected cells (Figure 7Biii). These results suggest that nuclear Akt can regulate, at least in part, hepatic Cx32.


Figure 7
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  		Fig. 7 Effect of Akt localization in MH1C1, MH1C1–Akt-WT+ and MH1C1–Akt-NES+ cells on Cx32. (A) MH1C1 transfected with cells using Akt-WT–GFP (I) and Akt-NES–GFP (II) expression vectors pEGFP-C1. Nuclei were stained using DAPI and examined under a fluorescent microscope (III and IV). The localization of Akt was analyzed using the ImagePro Plus computer software (V and VI) (Media Cybernetics, Silver Spring, MD). (B and C) MH1C1 transfected with cells using Akt-NES–GFP and Akt-WT–GFP expression vectors pEGFP-C1, and incubated with a mouse anti-Cx32 antibody (Sigma-Aldrich) and a goat anti-mouse-phycoerythrin antibody (Jackson Laboratories). Only positive events in GFP staining (red quadrant in ‘i’) were used to analyze Cx32 protein levels (ii). Cx32 expression was quantified by flow cytometry (50 000 events) with a FACSCalibur system (iii). b, P < 0.05, indicates a significant difference from ‘a’.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
We have previously shown that HCB treatment in female rats decreases hepatic Cx32 and Cx26 levels (8). Furthermore, we have also shown both in vivo and in vitro that the hepatic ILK pathway is activated by HCB treatment in female rats, and that this activation results in a decrease in the expression of E-cadherin (5). This decrease appears to result from the inactivation of GSK3ß, a downstream target of ILK. However, Cx32 levels were not affected by the inactivation of GSK3ß in vitro, suggesting that HCB-induced Cx32 downregulation occurs either downstream of ILK via the activation of a second sub-pathway, or by another unidentified parallel signaling pathway (5). Consequently, the objective of this study was to assess whether or not the activation of the ILK pathway is implicated in hepatic Cx32 regulation, and if so, to identify the sub-pathways implicated in this regulation. In this study, we have demonstrated that the overexpression of ILK in MH1C1 results in the activation and nuclear translocation of Akt, and that this activation is associated with a decrease in Cx32 mRNA levels. Combined with our previous results, the present data suggested that the activation of the ILK pathway by HCB results in the downregulation of both E-cadherin and Cx32, although this appears to be regulated via two different downstream targets, GSK3ß and Akt, respectively.

ILK is an evolutionarily conserved protein kinase, implicated in both integrin and growth-factor signaling. ILK basal activity is usually low, but can be stimulated by cell–cell interactions as well as by certain growth factors, including the epidermal growth factor receptor (EGFR) (12,13). Interestingly, it has been shown that HCB can stimulate EGFR (14,15). Stimulation of EGFR can activate the ILK pathway and consequently the PI3K pathway, which is associated with the recruitment of Akt to the plasma membrane (16). Whether or not EGFR or PI3K are implicated in ILK and Akt activation leading to the modulation of cell–cell interaction by HCB in female rats remains to be elucidated.

The role of the ILK pathway in the regulation of Cxs is not well defined. In another study it has been suggested that the loss of ß1 integrin function and inhibition of integrin clustering upregulates the expression of Cxs in embryonic stem (ES) cell-derived cardiomyocytes by ß-catenin/Wnt-dependent pathways (17). Downregulation of both adhering and gap junctions have been reported in certain malignant tumors, suggesting the existence of common pathways regulating the expression of proteins comprising these intercellular junctions (4,18). This is the first study to demonstrate a pathway by which both gap and adhering junction proteins are downregulated. Moreover, since both ILK pathway activation and decreased cell–cell interactions are hallmarks of cancer, our results suggest that this mechanism may represent a general mechanism implicated in carcinogenesis.

Little is known about pathways implicated in the regulation of Cxs. Several studies have shown that certain Cxs can be post-transcriptionally regulated by phosphorylation at different steps in the assembly of gap junctions, such as intracellular trafficking, assembly, gating and degradation (19). Various kinases have been implicated in Cx phosphorylation, including the mitogen-activated protein kinase (MAPK) (20). Cx43 has been reported to be regulated by a p38-MAPK in Sertoli cells (21), as well as liver Cx32 following partial hepatectomy in rat (22). Glucocorticoids have also been shown to be important regulators of hepatic GJIC and Cx32 expression (23).

However, while phosphorylation events are important for the post-translational regulation of Cx32, few studies have focused on the involvement of signaling pathways in the transcriptional regulation of Cx32. In our previous studies, HCB exposure resulted in Cx32 downregulation and increased Akt translocation into the nucleus, suggesting that nuclear Akt might be important in Cx32 regulation, as well as in carcinogenesis (5,8). In this study, ILK overexpression induced the nuclear translocation of Akt and a decrease in Cx32 mRNA levels. In order to determine whether or not Akt was responsible for the lower Cx32 levels, we transfected cells with an Akt expression vector. The resulting decrease in Cx32 mRNA levels indicated that the Akt pathway was at least partly responsible for the lower Cx32 levels. To determine if nuclear Akt was responsible for the lower Cx32 levels, cells were transfected with an expression vector (Akt-NES) that produced a modified active Akt that is specifically targeted to the nucleus. Flow cytometric analysis indicated a decrease in Cx32 expression in MH1C1 cells transfected with the nuclear active only Akt-NES, as compared with either inactive Akt-WT transfected or untransfected cells. Whether or not these effects are the result of Akt acting as a transcription factor or via the activation or inactivation of other transcription factors in the nucleus remains to be established.

Interestingly, it has been shown that Akt can phosphorylate Foxa-2 (hepatocyte nuclear factor-3ß), a transcription factor from the forkhead family (24). This factor is a member of the same family as HNF-1, which has been associated with Cx32 regulation (25,26). It has also been suggested that Sp1 may play a role in Cx32 regulation, since the Cx32 promoter contains several Sp1 sites and DNA–protein complexes have been identified in shift assays (25,27,28). Sp1 is a member of the Sp family of transcription factor frequently found in promoters lacking a TATA box and in the promoter regions of housekeeping genes (29). Sp1 transcription factors have been associated with the stimulation of the rat Cx32 promoter by acting at several sites along the 5' flanking region of the gene (27). A recent study has reported that Akt is required for the induction of VEGF, a critical event in carcinogenesis, and that this activation involves the phosphorylation and binding of Sp1 to the VEGF promoter region (30). Whether or not Akt can modulate Cx32 transcriptional regulation by acting on HNF-1 or Sp1 remains to be elucidated.

Recent studies have suggested that the nuclear localization of Akt may be important in the process of carcinogenesis. Indeed, it has been shown that Akt is localized into the nucleus of human thyroid tumors, and allows cell migration in a Boyden chamber in vitro (9,31). Furthermore, levels of nuclear Akt increase with stages of tumor progression in prostate cancer (32). Our study suggests a pro-carcinogenic role of Akt in modulating a decreased Cx32 expression, an important hallmark of hepatocarcinogenesis.

In summary, we have demonstrated that the overexpression of ILK in rat hepatocytes leads to the activation of Akt and its nuclear translocation. Active Akt is implicated in Cx32 downregulation via its nuclear effects, although we cannot discount the possibility that cytoplasmic Akt also contributes to lowering levels of Cx32. Together with our previous studies, these data suggest that the activation of the ILK pathway decreases cell–cell interactions by decreasing both gap and adhering junctions, via activation of two parallel branches of the same pathway. Since the downregulation of junctional proteins as well as the overexpression of ILK are hallmarks of carcinogenesis, this suggests that this mechanism can not only be a widespread event in HCB-induced or chemically induced tumors but may also be implicated in different types of cancers.


   Acknowledgments
 
Guylaine Lassonde, Julie Dufresne, Marcel Desrosiers and Mary Gregory are thanked for their assistance. Drs Matthew D.Ringel (Ohio State University) and Shoukat Dedhar (University of British Columbia) are thanked for their gifts of expression vectors. I.P. is the recipient of NSERC and Fondation Armand-Frappier studentships. This study was supported by a grant from the Canadian Liver Foundation to M.C. and D.G.C. as well as an NSERC discovery grant to D.G.C.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 28, 2006; revised April 13, 2006; accepted April 17, 2006.


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I. Plante, D. G. Cyr, and M. Charbonneau
Sexual Dimorphism in the Regulation of Liver Connexin32 Transcription in Hexachlorobenzene-Treated Rats
Toxicol. Sci., March 1, 2007; 96(1): 47 - 57.
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