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Am J Physiol Gastrointest Liver Physiol 293: G327-G334, 2007. First published April 12, 2007; doi:10.1152/ajpgi.00093.2007
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LIVER AND BILIARY TRACT

Differential regulation of cyclin D1 and cell death by bile acids in primary rat hepatocytes

Rui E. Castro,1,2 Joana D. Amaral,1 Susana Solá,1 Betsy T. Kren,2 Clifford J. Steer,2,3 and Cecília M. P. Rodrigues1

1Centro de Patogénese Molecular, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal; and Departments of 2Medicine and 3Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, Minnesota

Submitted 21 February 2007 ; accepted in final form 11 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ursodeoxycholic (UDCA) and tauroursodeoxycholic (TUDCA) acids modulate apoptosis and regulate cell-cycle effectors, including cyclin D1. In contrast, deoxycholic acid (DCA) induces cell death and cyclin D1. In this study, we explored the role of cyclin D1 in DCA-induced toxicity and further elucidated the antiapoptotic function of UDCA and TUDCA in primary rat hepatocytes. Cells were incubated with DCA and with or without UDCA or TUDCA for 8–30 h. In addition, hepatocytes were transfected with either an adenovirus expressing cyclin D1 or with a cyclin D1 reporter plasmid with or without bile acids. Finally, cells were cotransfected with short interfering RNA targeting p53. Unlike DCA, both UDCA and TUDCA reduced cyclin D1 expression and transcriptional activation, confirming our previous DNA microarray data. Furthermore, UDCA and TUDCA prevented DCA-induced cyclin D1 and cell death. Cyclin D1 overexpression increased DCA-induced Bax translocation, cytochrome c release, and apoptosis. However, UDCA and TUDCA were less efficient at decreasing cyclin D1 levels as well as DCA-induced changes with overexpression. Finally, after p53 silencing, the effects of cyclin D1 overexpression were almost completely abrogated, whereas UDCA and TUDCA cytoprotective potential was reestablished. In conclusion, cyclin D1 is a relevant player in modulating apoptosis by bile acids, in part through a p53-dependent mechanism.

apoptosis; Bax; liver; p53

URSODEOXYCHOLIC ACID (UDCA) stimulates hepatobiliary secretion and inhibits liver cell apoptosis (24). We and others (3, 28, 30) have shown that UDCA and tauroursodeoxycholic acid (TUDCA) reduce the apoptotic threshold in several cell types through modulation of classical mitochondrial pathways.

The mechanisms by which UDCA and TUDCA target mitochondria are now beginning to unfold. UDCA has been shown to inhibit hydrophobic deoxycholic acid (DCA)-induced Bax translocation both in vivo and in vitro (29, 30). In addition, we have previously shown (36) that UDCA can specifically modulate the E2F-1/p53/Bax apoptotic pathway, abrogating E2F-1-induced p53 and p53-associated Bax expression. Furthermore, UDCA's antiapoptotic effects involve the activation of nuclear steroid receptors coupled with the inhibition of the E2F-1/p53/Bax pathway (35). More recently, we have shown that UDCA and TUDCA regulate gene transcription, specifically apoptosis-, cell cycle- and proliferation-related genes (4). Cyclin D1 was found to be significantly downregulated by both bile acids. Interestingly, UDCA has previously been suggested to have a chemopreventive role in a rodent model of colonic carcinogenesis (10) and to protect against the development of human colon cancer (38). Other studies (40) have demonstrated that cyclin D1 may be involved in these processes.

Cyclin D1 is well known for regulating the G1 phase of the cell cycle by binding to and stimulating the activities of Cdk4 or Cdk6 (13). This process leads to phosphorylation and inhibition of the retinoblastoma protein, which no longer binds to and inhibits members of the E2F transcription factor family. Active E2F-1 can transactivate the expression of genes that are involved in the S-phase progression of the cell cycle and subsequent DNA synthesis (13). Disruptions in the formation of the Cdk-cyclin complexes may exacerbate cell growth and proliferation or, inversely, cell death. In this regard, several tumor types display amplification and/or overexpression of the cyclin D1 gene (15). Interestingly, elevated cyclin D1 levels can also lead to growth suppression and apoptosis (12, 33). Studies in human breast cancer cell lines have shown that increased expression of cyclin D1 initiates apoptotic events (26). Interestingly, p53 was found to be essential for cyclin D1 overexpression-induced Bax activation. However, other studies showed that both wild-type and mutant p53-expressing cells were sensitive to induction of apoptosis by overexpression of cyclin D1 (12). Thus it appears that cyclin D1-induced apoptosis may occur through both p53-dependent and -independent mechanisms.

We have previously shown (4) that UDCA and TUDCA modulate cell cycle- and apoptosis-related genes in primary rat hepatocytes, and we identified cyclin D1 as a main target. Here we further explored the role of cyclin D1 in DCA-induced toxicity as well as UDCA and TUDCA antiapoptotic function in hepatocytes. Our results indicate that DCA-induced apoptosis is associated with cyclin D1-dependent Bax translocation through nontranscriptional p53-dependent mechanisms. In addition, inhibition of cyclin D1 by UDCA and TUDCA attenuated DCA-induced effects.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Hepatocyte cultures and bile-acid treatment. Rat primary hepatocytes were isolated from male Sprague-Dawley rats (100–150 g) by collagenase perfusion (19). After isolation, hepatocytes were resuspended in Williams' E medium (Sigma-Aldrich, St. Louis, MO) with supplements and 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA) (36). Cells were plated at either 5 x 104 cells/cm2 for viability assays and apoptosis detection or at 2.5 x 105 cells/cm2 for other experiments. Twenty-four hours after being plated, cells were treated with either 100 µM UDCA or TUDCA (Sigma-Aldrich) or no addition (control) for 1 h. Cells were then exposed to 50 µM DCA (Sigma-Aldrich) for an additional 8, 18, or 30 h. Bile-acid concentrations were selected based on previous cell-culture and animal studies and are the same order of magnitude as those found in patients with cholestatic liver disease undergoing UDCA therapy (24).

Measurement of cell death. Cell viability was measured by lactate dehydrogenase (LDH) viability assays (Sigma-Aldrich) using culture medium. In addition, Hoechst labeling of attached cells was used to detect apoptotic nuclei (36). Three random microscopic fields per sample of ~150 nuclei were counted, and mean values were expressed as the percentage of apoptotic nuclei. The terminal transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assays were performed according to the manufacturer's recommendations (Serological, Norcross, GA).

Cyclin D1 transfections and luciferase assays. Transfections were performed by using a cyclin D1 adenovirus expression construct and a cyclin D1 promoter-driven luciferase reporter. The recombinant cyclin D1 adenovirus (ADV-D1) was constructed by ligating the EcoRI/HindIII fragment of human cyclin D1 into pACCMV.pLpA (1). An identical construct containing the nuclear-localizing variant of beta-galactosidase (ADV-betagal) was used as control. The cyclin D1 promoter-driven luciferase construct consisted of a 1,745-bp human cyclin D1 promoter fused to the luciferase gene (25). Cells were infected with ADV-D1 or ADV-betagal 4 h after being plated at a dose of 20 plaque-forming units/cell. The medium containing the adenovirus was removed and replaced with virus-free medium after 2 h. Cells were treated with bile acids and were harvested for total, cytosolic, mitochondrial, and nuclear protein extraction and immunoblotting. Attached cells were also fixed for morphological detection of apoptosis.

In parallel experiments, 12 h after being plated, hepatocytes were transfected with 4 µg of cyclin D1 promoter-driven luciferase reporter by using conjugated polyethylenimine (16). For normalization, cells were cotransfected with the chloramphenicol acetyltransferase (CAT) reporter construct, pCAT3-Control (Promega, Madison, WI). Transfection efficiencies of ~70% were determined by using a reporter plasmid expressing beta-galactosidase and did not differ between reporter and expression plasmids (data not shown). Cells were treated with bile acids and were maintained in culture for additional 8, 18, or 30 h, after which they were harvested for CAT ELISA (Roche Applied Science, Indianapolis, IN) and luciferase assays (Promega).

Short interference-mediated silencing of the p53 gene. A pool of three target-specific, 20- to 25-nt short interfering RNAs (siRNAs) was used to knock down p53 gene expression in rats (Santa Cruz Biotechnology, Santa Cruz, CA). Control siRNAs containing a scrambled sequence that does not lead to the specific degradation of any known cellular mRNA were used as control. Four hours after being plated, hepatocytes were transfected by using JetSI for siRNA (Polyplus Transfections, Illkirch, France). Four hours later, cells were transfected with ADV-D1 and were treated with bile acids. Floating and attached cells were harvested for total, cytosolic, and mitochondrial protein extraction followed by immunoblotting. Attached cells were fixed for Hoechst staining, and culture medium was collected for LDH assays.

Immunoblotting. Cellular distributions of Bax and cytochrome c were determined by using mitochondrial and cytosolic protein extracts as previously described (5). Forty micrograms of mitochondrial and cytosolic proteins were separated by 15% SDS-PAGE. Blots were incubated with a primary monoclonal antibody to either Bax (Santa Cruz Biotechnology) or cytochrome c (PharMingen, San Diego, CA). Mitochondrial contamination of the cytosolic protein extracts was determined by Western blot analysis of cytochrome c oxidase (subunit II). In addition, 10–50 µg of total protein extracts were separated on 10% SDS-PAGE, and blots were probed with either primary mouse monoclonal antibodies reactive with p53 and Bax or primary rabbit polyclonal antibodies to cyclin D1 (Santa Cruz Biotechnology). beta-Actin and cytochrome c oxidase (subunit II) were used as loading controls.

Densitometry and statistical analysis. The relative intensities of protein bands were analyzed by using the Quantity One version 4.6 densitometric analysis program (Bio-Rad Laboratories). Statistical analysis was performed by using GraphPad InStat version 3.00 (GraphPad Software, San Diego, CA) for the analysis of variance and Bonferroni's multiple-comparison tests. Values of P < 0.05 were considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
UDCA and TUDCA reduce DCA-induced cell death and cyclin D1 expression. In addition to its role in cell proliferation and oncogenesis, cyclin D1 is also involved in apoptosis (13). Furthermore, using microarray analysis, we (4) have previously identified cyclin D1 as a novel potential target of UDCA and TUDCA in primary rat hepatocytes. Therefore, we hypothesized that cyclin D1 may play a critical role in bile acid-induced modulation of apoptosis and cell survival.

In this study, incubation of primary rat hepatocytes with DCA resulted in significant levels of general cell death (Fig. 1A) and apoptosis (P < 0.05, Fig. 1B). Annexin V/propidium iodide double staining further confirmed these results (data not shown). In contrast, UDCA and TUDCA inhibited DCA-induced cell death by ~50% (P < 0.05) throughout the time course (Fig. 1, A and B). Similar results were obtained when apoptosis was assessed by TUNEL staining (Fig. 1C).


Figure 1
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  		Fig. 1. Ursodeoxycholic (UDCA) and tauroursodeoxycholic (TUDCA) acids inhibit cell death induced by deoxycholic acid (DCA) in primary rat hepatocytes. Cells were incubated with 50 µM DCA or no addition (control) in the presence or absence of UDCA or TUDCA for 8, 18, and 30 h. In coincubation experiments, cells were pretreated with either 100 µM UDCA or TUDCA 1 h before incubation with DCA. Cells were fixed and stained for morphological evaluation of apoptosis and terminal transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assays. A: culture medium was used for lactate dehydrogenase (LDH) viability assays. Results are expressed as means ± SE of fold change from control. B, top: fluorescent microscopy of Hoechst staining in control hepatocytes (a) and in cells exposed to either DCA (b), UDCA (c), DCA + UDCA (d), TUDCA (e), or DCA + TUDCA (f) for 18 h. Normal nuclei showed noncondensed chromatin dispersed over the entire nucleus. Apoptotic nuclei were identified by condensed chromatin, contiguous to the nuclear membrane, as well as nuclear fragmentation of condensed chromatin (arrows). Bottom: percent apoptosis of cells exposed to DCA in the presence or absence of UDCA or TUDCA for the indicated time points. Results are expressed as means ± SE of percentage of apoptotic nuclei. C, top: TUNEL staining in control hepatocytes (a) and in cells exposed to either DCA (b), UDCA (c), DCA + UDCA (d), TUDCA (e), or DCA + TUDCA (f) for 18 h. Apoptotic cells were identified by condensed nucleus with brownish staining (arrows); normal cells were counterstained with methyl green. Bottom: percent apoptosis of cells incubated with DCA in the presence or absence of UDCA or TUDCA for 18 h. Results are expressed as means ± SE of percentage of apoptotic nuclei. *P < 0.01 from control; {dagger}P < 0.05 and {ddagger}P < 0.01 from DCA alone.

 
Cyclin D1 protein increased above control after 18 h of incubation with DCA (P < 0.05; Fig. 2A). UDCA and TUDCA alone decreased cyclin D1 protein (P < 0.05; Fig. 2B) in agreement with reduced cyclin D1 mRNA transcripts in the microarray analysis. Importantly, UDCA and TUDCA abrogated DCA-induced cyclin D1 expression (P < 0.01 and P < 0.05, respectively). Semiquantitative RT-PCR showed a twofold increase in cyclin D1 gene by DCA (P < 0.05), which was inhibited by ~50% after preincubation with UDCA or TUDCA (data not shown). Furthermore, DCA also induced cyclin D1 promoter activity by ~2.5- and 3-fold at 8 and 18 h of incubation, respectively (P < 0.05; Fig. 2C). In agreement with the mRNA and protein-expression data, UDCA and TUDCA alone significantly decreased cyclin D1 promoter activity (P < 0.05) and DCA-mediated changes in cyclin D1 expression levels (P < 0.05).


Figure 2
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  		Fig. 2. Cyclin D1 transcription and expression is modulated by bile acids in primary rat hepatocytes. Cells were incubated with 50 µM DCA or no addition (control) for 8, 18, and 30 h or were coincubated with UDCA or TUDCA. Cells were pretreated with 100 µM UDCA or TUDCA 1 h before incubation with DCA for 8 or 18 h. Total proteins were extracted for Western blot analysis. In addition, 12 h before addition of DCA, cells were cotransfected with a cyclin D1 promoter-driven luciferase construct and a chloramphenicol acetyltransferase (CAT) reporter control plasmid as described in MATERIALS AND METHODS. A: representative immunoblot of cyclin D1 and corresponding histogram in cells incubated with DCA for the indicated time points. B: representative immunoblot of cyclin D1 and corresponding histogram in cells incubated with DCA alone or in the presence or absence of UDCA or TUDCA for 18 h. Blots were normalized to endogenous beta-actin. C: cyclin D1 promoter activity in cells incubated with DCA for the indicated time points. D: cyclin D1 promoter activity in cells incubated with DCA alone or in the presence or absence of UDCA or TUDCA for 8 h. Cyclin D1 promoter-driven luciferase activity (absorbency/mg protein) was normalized to control CAT expression. Results are expressed as means ± SE of arbitrary units of at least 3 different experiments. §P < 0.05 from control; {dagger}P < 0.05 and {ddagger}P < 0.01 from DCA alone.

 
Cyclin D1 expression is involved in modulation of apoptosis. On the basis of the information that bile acids differentially regulate cyclin D1 expression levels, we next investigated whether cyclin D1 contributed to modulation of apoptosis. ADV-D1 was transfected into cells, and immunoblot analysis showed that ADV-D1 transfection resulted in high-level expression of cyclin D1 (>20-fold) compared with cyclin D1 levels in ADV-betagal-infected cells (Fig. 3A). Freshly isolated primary hepatocytes are primarily in the G0 phase of the cell cycle. In the presence of growth factors, they slowly progress through G1 phase, entering S phase 42–45 h after being plated (1). To minimize the effects of endogenous cyclin D1 or other cell-cycle factors in our studies, overexpression experiments were performed during the first 30 h after hepatocyte isolation. At this time, cells are in mid-G1 phase (27) and produce relatively low levels of cyclin D1 (Fig. 3A).


Figure 3
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  		Fig. 3. Cyclin D1 expression is involved in modulation of apoptosis by bile acids. Cells were incubated with 50 µM DCA or no addition (control) in the presence or absence of UDCA or TUDCA for 8 h. In coincubation experiments, cells were pretreated with 100 µM UDCA or TUDCA 1 h before incubation with DCA. Cells were infected with cyclin D1 adenovirus (ADV-D1) or control beta-galactosidase adenovirus (ADV-betagal) 20 h before addition of DCA. Cells were fixed and stained for morphological evaluation of apoptosis. In addition, total proteins were extracted and subjected to Western blot analysis of cyclin D1 as described in MATERIALS AND METHODS. A: representative immunoblots of cyclin D1 in cells infected with ADV-betagal and ADV-D1 for 28 h and cells infected with ADV-betagal for the indicated times. B: percent apoptosis of cells exposed to bile acids and infected with ADV-betagal or ADV-D1. C: representative immunoblots of cyclin D1 and corresponding histograms in cells incubated with bile acids and infected with ADV-betagal or ADV-D1. ADV-D1 infection led to a >20-fold increase of cyclin D1 compared with endogenous cyclin D1 levels assayed in ADV-betagal-infected cells. Blots were normalized to endogenous beta-actin, and results are expressed as means ± SE of arbitrary units of at least 3 different experiments. §P < 0.05 and *P < 0.01 from control; {dagger}P < 0.05 and {ddagger}P < 0.01 from DCA alone.

 
Surprisingly, cyclin D1 overexpression only slightly increased basal apoptosis; however, it significantly augmented the ability of DCA to induce apoptosis (P < 0.01; Fig. 3B). Decreasing cyclin D1 levels by culturing cells in the absence of serum did not significantly reduce DCA-induced cell death (data not shown), suggesting that other factors may play a role. UDCA and TUDCA alone induced a moderate degree of apoptosis in cyclin D1-overexpressing cells (P < 0.01). In addition, UDCA and TUDCA were ~50% less efficient at reducing DCA-induced apoptosis when cyclin D1 was overexpressed. Similarly, the ability of UDCA and TUDCA to decrease cyclin D1 expression alone or in combination with DCA was significantly impaired when the protein was overexpressed by the adenovirus (Fig. 3C). These results suggest that downregulation of cyclin D1 is important in UDCA and TUDCA promotion of cell survival.

Bax translocation and cytochrome c release are amplified by cyclin D1. It was important to better define the mechanisms of apoptosis in hepatocytes with bile acids and/or cyclin D1 overexpression. Although not statistically significant, total Bax protein levels were slightly increased as a result of DCA incubation in both ADV-betagal- and ADV-D1-infected cells (Fig. 4A). Nevertheless, our results indicated that DCA induced Bax translocation (P < 0.05), which in turn was prevented by UDCA and TUDCA (P < 0.05; Fig. 4B). Surprisingly, cyclin D1 overexpression resulted in Bax translocation when compared with ADV-betagal-expressing cells (P < 0.05). DCA treatment additionally increased Bax translocation in cyclin D1-infected hepatocytes. Importantly, UDCA and TUDCA were less efficient at protecting against DCA's effects. Data on cytochrome c release into the cytosol were in agreement with Bax changes (Fig. 4B).


Figure 4
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  		Fig. 4. Bile-acid effects on Bax translocation and cytochrome c release are influenced by cyclin D1. Cells were incubated with 50 µM DCA or no addition (control) in the presence or absence of UDCA or TUDCA for 8 h. In coincubation experiments, cells were pretreated with 100 µM UDCA or TUDCA 1 h before incubation with DCA. Cells were infected with ADV-D1 or ADV-betagal 20 h before addition of DCA. Total, mitochondrial, and cytosolic protein fractions were extracted for Western blot analysis as described in MATERIALS AND METHODS. Fold changes were calculated on the basis of densitometry analysis of at least 3 independent experiments. Representative immunoblots are shown. A: total Bax. Blots were normalized to endogenous beta-actin. B: mitochondrial Bax and supernatant cytochrome c. Blots were normalized to cytochrome c oxidase II (Cox II) or beta-actin.

 
Cyclin D1-induced Bax translocation is p53 dependent. It is thought that cyclin D1-induced apoptosis occurs through either p53-dependent or -independent mechanisms (13). p53, in turn, may induce apoptosis by modulating multiple pro- and antiapoptotic gene products (41) and Bax translocation to the mitochondrial membrane (11). In addition, we have previously shown (36) that UDCA specifically inhibits the E2F-1/p53/Bax apoptotic pathway in primary rat hepatocytes. Neither p53 total levels nor nuclear trafficking were significantly affected by bile acids compared with controls. Furthermore and as predicted, p53 expression levels under basal conditions or following bile acid incubation were very low (data not shown). Nevertheless, to further investigate the role of p53, primary rat hepatocytes were transfected with p53 siRNA and infected with either ADV-betagal or ADV-D1 in the presence or absence of bile acids. By Western blot analysis, p53 siRNA suppressed expression of p53 by ~65% compared with control cells transfected with an irrelevant siRNA (Fig. 5A). Furthermore, cyclin D1 protein levels were slightly reduced in ADV-D1-infected hepatocytes with silencing. Nevertheless, modulation of cyclin D1 by bile acids was not significantly affected by p53 siRNA transfections in both ADV-betagal- and ADV-D1-infected cells (data not shown). Apoptosis was markedly reduced in p53 siRNA-transfected cells (Fig. 5B). In addition, cyclin D1 overexpression no longer decreased UDCA and TUDCA inhibition of DCA-induced apoptosis after p53 gene silencing. Similar results were obtained by using LDH viability assays (data not shown). Thus p53 plays a key role during cyclin D1-mediated apoptosis by bile acids. Interestingly, Bax translocation by bile acids was also inhibited and was not significantly different between ADV-betagal and ADV-D1-infected cells following transfection with p53 siRNA (Fig. 5C). Inhibition of Bax translocation in p53 siRNA-transfected cells did not completely inhibit apoptosis induced by DCA, suggesting that additional mechanisms contribute to apoptosis by bile acids.


Figure 5
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  		Fig. 5. Cyclin D1-induced translocation of Bax is p53 dependent. Cells were incubated with 50 µM DCA or no addition (control) in the presence or absence of UDCA or TUDCA for 8 h. In coincubation experiments, cells were pretreated with 100 µM UDCA or TUDCA 1 h before incubation with DCA. Cells were infected with ADV-D1 or ADV-betagal 16 h before addition of DCA. In addition, cells were transfected with either control or p53 siRNA 4 h before adenoviral infection. Cells were fixed and stained for morphological evidence of apoptosis. In addition, total proteins were extracted and subjected to Western blot analysis of p53 and cyclin D1. Total, mitochondrial, and cytosolic proteins were isolated for Bax analysis as described in MATERIALS AND METHODS. Fold changes were calculated based on densitometry analysis of at least 3 independent experiments. Representative immunoblots are shown. A: p53 and cyclin D1. B: percentage of apoptosis. C: total and mitochondrial Bax in cells incubated with bile acids and transfected with p53 siRNA in the presence or absence of ADV-betagal or ADV-D1 (top) and histogram of Bax mitochondrial levels in cells incubated with bile acids and transfected with control or p53 siRNA in the presence or absence of ADV-betagal or ADV-D1 (bottom). Blots were normalized to cytochrome c oxidase II (Cox II) or beta-actin. Results are expressed as means ± SE of arbitrary units of at least 3 different experiments. {dagger}P < 0.05 and {ddagger}P < 0.01 from control; *P < 0.01 from corresponding control siRNA-transfected cells.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The mechanisms of bile acid effects on cell survival and apoptosis are not entirely understood. We have previously reported that UDCA is able to prevent apoptosis in primary rat hepatocytes by inhibiting the mitochondrial pathway (28, 30) and modulating the E2F-1/p53/Bax apoptotic cascade (36). In addition, we have recently shown that UDCA and TUDCA specifically regulate cell cycle-related genes, including cyclin D1 (4). Hydrophobic bile acids such as DCA may either promote cancer growth or induce cell death and apoptosis (21). DCA increases cyclin D1 mRNA and protein expression in both primary rat hepatocytes and colon cancer cell lines (17, 23). Our results address the mechanisms by which bile acids modulate apoptosis upstream of mitochondrial commitment and demonstrate that cyclin D1 facilitates apoptosis of primary rat hepatocytes and is modulated differentially by bile acids. In fact, DCA exposure resulted in cyclin D1 expression and cell death, but preincubation with UDCA and TUDCA significantly abrogated both effects.

The role of cyclin D1 as a facilitator of hepatocyte apoptosis was further established by cyclin D1 overexpression. Previous studies have already suggested that increased expression of cyclin D1 initiates apoptotic events that are, however, incapable of culminating in cell death (26). In fact, cyclin D1 overexpression per se only marginally increased basal levels of apoptosis but significantly amplified DCA-induced apoptosis while decreasing UDCA and TUDCA cytoprotection. Our results suggested that UDCA and TUDCA downregulate cyclin D1 expression at the transcriptional level, possibly explaining their reduced antiapoptotic properties after cyclin D1 overexpression. Importantly, although significantly reduced, the antiapoptotic properties of UDCA and TUDCA were not completely abolished, indicating that they are only partially dependent on cyclin D1.

Although the role of cyclin D1 in proliferation is well established, less is known about its function in promoting apoptosis. It is thought that cyclin D1-induced apoptosis may occur through several distinct mechanisms (13). Elevated cyclin D1 levels could eliminate retinoblastoma protein function through its functional inactivation, releasing free E2F-1, which directly or in combination with other proteins may activate genes necessary for apoptosis (9). In that sense, we have previously shown that TGF-beta1-induced apoptosis is associated with activation of E2F-1, which in turn is inhibited by UDCA (36). Other studies (22, 26) suggest that cyclin D1 may also amplify stress-induced mitochondrial pathways of apoptosis. Here we confirmed that UDCA and TUDCA prevented DCA-induced Bax translocation and cytochrome c release. Interestingly, cyclin D1 overexpression amplified DCA-induced Bax translocation and cytochrome c release and reduced UDCA and TUDCA cytoprotection. In addition, cyclin D1 overexpression alone increased Bax translocation and cytochrome c, without significantly altering apoptosis. Similar results have been reported in other models of cyclin D1 overexpression (26) and, although not completely understood, they suggest that in the absence of an additional toxic stimuli, endogenous levels of Bcl-2 antiapoptotic proteins are sufficient to inhibit cell death, even at a postmitochondrial level (8, 32). Alternatively, mitochondrial function may not be severely impaired in such cases (20).

Previous findings have shown that UDCA and TUDCA modulate the E2F-1/p53/Bax apoptotic pathway (34, 36). Thus we investigated whether modulation of cyclin D1-associated apoptosis by bile acids involved the p53 protein. Here we show that p53 total and nuclear levels were very low and only slightly modulated by either bile acids or cyclin D1 overexpression, stressing the importance of UDCA's and TUDCA's effects on mitochondria, as previously described (28). Importantly, modulation of apoptosis by DCA through cyclin D1 appears to depend on p53. Gene silencing of p53 completely abrogated cyclin D1's effects in Bax translocation, cytochrome c release, and apoptosis, despite the elevated levels of cyclin D1. Although the precise mechanism remains unclear, it conceivably involves increased cyclin D1 translocation to the nucleus or, alternatively, inhibition of its nuclear export (37). Although moderate levels of nuclear cyclin D1 are usually responsible for cell-cycle progression, higher levels may generate sufficient genetic instability that results in activation of p53 and apoptosis. With transactivation of cyclin D1, DCA may be amplifying the latter effect, whereas UDCA and TUDCA are effective at inhibiting it. Interestingly, we have shown that UDCA reduces E2F-1 transcriptional activation and p53 stabilization induced by TGF-beta1 (36). Therefore, cyclin D1 may represent an alternative, upstream target of this pathway by activating E2F-1.

In addition, hepatocytes were still able to undergo DCA-induced apoptosis in the absence of p53, although to a lesser extent. In fact, p53 gene silencing significantly inhibited overall Bax translocation to the mitochondria. This implies the existence of both p53-dependent and -independent signals modulated by bile acids, and in fact Fas-mediated signaling, for example, is thought to be involved in bile-acid induced liver injury (14).

The mechanism by which p53 modulates cyclin D1-induced Bax translocation and apoptosis remains to be determined, because p53 protein levels were relatively unchanged. Nevertheless, p53 is highly governed through complex networks of posttranslational modifications (39) and may even promote cell death by direct interaction with the mitochondria (18, 31). This may result in the activation of Bax in the cytoplasm (7).

Finally, p53 is known to transcriptionally activate several p53-responsive genes, including Noxa and Puma (2), which may act in a Bid-like fashion to facilitate Bax activation. The induction of cyclin D1 can additionally be mediated by a target of p53, the p21 (WAF1/CIP1) inhibitor of cyclin-dependent kinases (6). In this regard, it was recently shown that p21 potentiates bile acid-induced p53-dependent apoptosis in hepatocytes (27). Therefore, further studies are necessary to determine whether modulation of Puma, Noxa, p21, or other proteins are responsible for cyclin D1 amplification of bile acid-induced apoptosis. These studies would also benefit our current understanding about bile acid function in cell proliferation. In fact, the (patho)physiological significance of our data may be more relevant considering different genetic backgrounds. DCA has been shown to use the beta-catenin-cyclin D1 pathway to promote colon cancer (23). On the other hand, UDCA has been shown to have a chemopreventive role in a rat model of colon cancer by inhibiting azoxymethane-induced cyclin D1 (40). Therefore, cyclin D1 may provide the link between the apparently conflicting roles of bile acids in cell proliferation and apoptosis.

Collectively, our studies suggest that DCA-induced mitochondrial apoptosis is associated with and amplified by cyclin D1. UDCA and TUDCA inhibition of cyclin D1 expression contribute to their antiapoptotic effects. Furthermore, bile acid modulation of apoptosis through cyclin D1 is associated with p53-dependent Bax translocation to the mitochondria. The identification and characterization of upstream apoptotic targets for bile acids may result in the development of novel therapeutic options for diseases associated with deregulated apoptosis.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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REFERENCES
 
This work was supported by grants POCI/SAU-FCF/62479/2004 from Fundação para a Ciência e a Tecnologia (FCT), Lisbon, Portugal (to C. H. P. Rodrigues). R. E. Castro and J. D. Amaral were recipients of Ph.D. fellowships (BD/12655/2003 and BD/17799/2004, respectively), and S. Solá was recipient of postdoctoral fellowship (BPD/20834/2004) from FCT.


   ACKNOWLEDGMENTS
 
We thank Dr. Jeffrey H. Albrecht, University of Minnesota (Minneapolis, MN) for providing the cyclin D1 adenovirus and Dr. Richard Pestell, Jefferson Medical College (Philadelphia, PA) for the cyclin D1 promoter-driven luciferase gene. We also thank all members of the laboratory for continual support and encouragement.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. M. P. Rodrigues, Centro de Patogénese Molecular, Faculty of Pharmacy, Univ. of Lisbon, Av. das Forças Armadas, 1600-083 Lisbon, Portugal (e-mail: cmprodrigues{at}ff.ul.pt)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 GRANTS
 REFERENCES
 

  1. Albrecht JH, Hansen LK. Cyclin D1 promotes mitogen-independent cell cycle progression in hepatocytes. Cell Growth Differ 10: 397–404, 1999.[Abstract/Free Full Text]
  2. Balint EE, Vousden KH. Activation and activities of the p53 tumour suppressor protein. Br J Cancer 85: 1813–1823, 2001.[CrossRef][Web of Science][Medline]
  3. Botla R, Spivey JR, Aguilar H, Bronk SF, Gores GJ. Ursodeoxycholate (UDCA) inhibits the mitochondrial membrane permeability transition induced by glycochenodeoxycholate: a mechanism of UDCA cytoprotection. J Pharmacol Exp Ther 272: 930–938, 1995.[Abstract/Free Full Text]
  4. Castro RE, Solá S, Ma X, Ramalho RM, Kren BT, Steer CJ, Rodrigues CMP. A distinct microarray gene expression profile in primary rat hepatocytes incubated with ursodeoxycholic acid. J Hepatol 42: 897–906, 2005.[CrossRef][Web of Science][Medline]
  5. Castro RE, Solá S, Ramalho RM, Steer CJ, Rodrigues CMP. The bile acid tauroursodeoxycholic acid modulates phosphorylation and translocation of bad via phosphatidylinositol 3-kinase in glutamate-induced apoptosis of rat cortical neurons. J Pharmacol Exp Ther 311: 845–852, 2004.[Abstract/Free Full Text]
  6. Chen X, Bargonetti J, Prives C. p53, through p21 (WAF1/CIP1), induces cyclin D1 synthesis. Cancer Res 55: 4257–4263, 1995.[Abstract/Free Full Text]
  7. Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Schuler M, Green DR. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science 303: 1010–1014, 2004.[Abstract/Free Full Text]
  8. Conus S, Rosse T, Borner C. Failure of Bcl-2 family members to interact with Apaf-1 in normal and apoptotic cells. Cell Death Differ 7: 947–954, 2000.[CrossRef][Web of Science][Medline]
  9. Duquesne F, Florent M, Roue G, Troussard X, Sola B. Ectopic expression of cyclin D1 impairs the proliferation and enhances the apoptosis of a murine lymphoid cell line. Cell Death Differ 8: 51–62, 2001.[CrossRef][Web of Science][Medline]
  10. Earnest DL, Holubec H, Wali RK, Jolley CS, Bissonette M, Bhattacharyya AK, Roy H, Khare S, Brasitus TA. Chemoprevention of azoxymethane-induced colonic carcinogenesis by supplemental dietary ursodeoxycholic acid. Cancer Res 54: 5071–5074, 1994.[Abstract/Free Full Text]
  11. Gao CF, Ren S, Zhang L, Nakajima T, Ichinose S, Hara T, Koike K, Tsuchida N. Caspase-dependent cytosolic release of cytochrome c and membrane translocation of Bax in p53-induced apoptosis. Exp Cell Res 265: 145–151, 2001.[CrossRef][Web of Science][Medline]
  12. Han EK, Begemann M, Sgambato A, Soh JW, Doki Y, Xing WQ, Liu W, Weinstein IB. Increased expression of cyclin D1 in a murine mammary epithelial cell line induces p27kip1, inhibits growth, and enhances apoptosis. Cell Growth Differ 7: 699–710, 1996.[Abstract]
  13. Han EK, Ng SC, Arber N, Begemann M, Weinstein IB. Roles of cyclin D1 and related genes in growth inhibition, senescence and apoptosis. Apoptosis 4: 213–219, 1999.[CrossRef][Web of Science][Medline]
  14. Higuchi H, Gores GJ. Bile acid regulation of hepatic physiology. IV. Bile acids and death receptors. Am J Physiol Gastrointest Liver Physiol 284: G734–G738, 2003.[Abstract/Free Full Text]
  15. Hunter T, Pines J. Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age. Cell 79: 573–582, 1994.[CrossRef][Web of Science][Medline]
  16. Kren BT, Cole-Strauss A, Kmiec EB, Steer CJ. Targeted nucleotide exchange in the alkaline phosphatase gene of HuH-7 cells mediated by a chimeric RNA/DNA oligonucleotide. Hepatology 25: 1462–1468, 1997.[CrossRef][Web of Science]
  17. Kren BT, Rodrigues CMP, Setchell KD, Steer CJ. Posttranscriptional regulation of mRNA levels in rat liver associated with deoxycholic acid feeding. Am J Physiol Gastrointest Liver Physiol 269: G961–G973, 1995.[Abstract/Free Full Text]
  18. Marchenko ND, Zaika A, Moll UM. Death signal-induced localization of p53 protein to mitochondria. A potential role in apoptotic signaling. J Biol Chem 275: 16202–16212, 2000.[Abstract/Free Full Text]
  19. Mariash CN, Seelig S, Schwartz HL, Oppenheimer JH. Rapid synergistic interaction between thyroid hormone and carbohydrate on mRNAS14 induction. J Biol Chem 261: 9583–9586, 1986.[Abstract/Free Full Text]
  20. Martinou JC, Desagher S, Antonsson B. Cytochrome c release from mitochondria: all or nothing. Nat Cell Biol 2: E41–E43, 2000.[CrossRef][Web of Science][Medline]
  21. Milovic V, Teller IC, Faust D, Caspary WF, Stein J. Effects of deoxycholate on human colon cancer cells: apoptosis or proliferation. Eur J Clin Invest 32: 29–34, 2002.[CrossRef][Web of Science][Medline]
  22. Niu MY, Menard M, Reed JC, Krajewski S, Pratt MA. Ectopic expression of cyclin D1 amplifies a retinoic acid-induced mitochondrial death pathway in breast cancer cells. Oncogene 20: 3506–3518, 2001.[CrossRef][Web of Science][Medline]
  23. Pai R, Tarnawski AS, Tran T. Deoxycholic acid activates beta-catenin signaling pathway and increases colon cell cancer growth and invasiveness. Mol Biol Cell 15: 2156–2163, 2004.[Abstract/Free Full Text]
  24. Paumgartner G, Beuers U. Ursodeoxycholic acid in cholestatic liver disease: mechanisms of action and therapeutic use revisited. Hepatology 36: 525–531, 2002.[CrossRef][Web of Science][Medline]
  25. Pestell RG, Albanese C, Watanabe G, Johnson J, Eklund N, Lastowiecki P, Jameson JL. Epidermal growth factor and c-Jun act via a common DNA regulatory element to stimulate transcription of the ovine P-450 cholesterol side chain cleavage (CYP11A1) promoter. J Biol Chem 270: 18301–18308, 1995.[Abstract/Free Full Text]
  26. Pratt MA, Niu MY. Bcl-2 controls caspase activation following a p53-dependent cyclin D1-induced death signal. J Biol Chem 278: 14219–14229, 2003.[Abstract/Free Full Text]
  27. Qiao L, McKinstry R, Gupta S, Gilfor D, Windle JJ, Hylemon PB, Grant S, Fisher PB, Dent P. Cyclin kinase inhibitor p21 potentiates bile acid-induced apoptosis in hepatocytes that is dependent on p53. Hepatology 36: 39–48, 2002.[CrossRef][Web of Science][Medline]
  28. Rodrigues CMP, Fan G, Ma X, Kren BT, Steer CJ. A novel role for ursodeoxycholic acid in inhibiting apoptosis by modulating mitochondrial membrane perturbation. J Clin Invest 101: 2790–2799, 1998.[Web of Science][Medline]
  29. Rodrigues CMP, Fan G, Wong PY, Kren BT, Steer CJ. Ursodeoxycholic acid may inhibit deoxycholic acid-induced apoptosis by modulating mitochondrial transmembrane potential and reactive oxygen species production. Mol Med 4: 165–178, 1998.[CrossRef][Web of Science][Medline]
  30. Rodrigues CMP, Ma X, Linehan-Stieers C, Fan G, Kren BT, Steer CJ. Ursodeoxycholic acid prevents cytochrome c release in apoptosis by inhibiting mitochondrial membrane depolarization and channel formation. Cell Death Differ 6: 842–854, 1999.[CrossRef][Web of Science][Medline]
  31. Schuler M, Bossy-Wetzel E, Goldstein JC, Fitzgerald P, Green DR. p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release. J Biol Chem 275: 7337–7342, 2000.[Abstract/Free Full Text]
  32. Shimizu S, Eguchi Y, Kamiike W, Funahashi Y, Mignon A, Lacronique V, Matsuda H, Tsujimoto Y. Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating proton flux. Proc Natl Acad Sci USA 95: 1455–1459, 1998.[Abstract/Free Full Text]
  33. Sofer-Levi Y, Resnitzky D. Apoptosis induced by ectopic expression of cyclin D1 but not cyclin E. Oncogene 13: 2431–2437, 1996.[Web of Science][Medline]
  34. Solá S, Amaral JD, Castro RE, Ramalho RM, Borralho PM, Kren BT, Tanaka H, Steer CJ, Rodrigues CMP. Nuclear translocation of UDCA by the glucocorticoid receptor is required to reduce TGF-beta1-induced apoptosis in rat hepatocytes. Hepatology 42: 925–934, 2005.[CrossRef][Web of Science]
  35. Solá S, Castro RE, Kren BT, Steer CJ, Rodrigues CMP. Modulation of nuclear steroid receptors by ursodeoxycholic acid inhibits TGF-beta1-induced E2F-1/p53-mediated apoptosis of rat hepatocytes. Biochemistry 43: 8429–8438, 2004.[CrossRef][Medline]
  36. Solá S, Ma X, Castro RE, Kren BT, Steer CJ, Rodrigues CMP. Ursodeoxycholic acid modulates E2F-1 and p53 expression through a caspase-independent mechanism in transforming growth factor beta1-induced apoptosis of rat hepatocytes. J Biol Chem 278: 48831–48838, 2003.[Abstract/Free Full Text]
  37. Sumrejkanchanakij P, Tamamori-Adachi M, Matsunaga Y, Eto K, Ikeda MA. Role of cyclin D1 cytoplasmic sequestration in the survival of postmitotic neurons. Oncogene 22: 8723–8730, 2003.[CrossRef][Web of Science][Medline]
  38. Tung BY, Emond MJ, Haggitt RC, Bronner MP, Kimmey MB, Kowdley KV, Brentnall TA. Ursodiol use is associated with lower prevalence of colonic neoplasia in patients with ulcerative colitis and primary sclerosing cholangitis. Ann Intern Med 134: 89–95, 2001.[Abstract/Free Full Text]
  39. Vousden KH, Lu X. Live or let die: the cell's response to p53. Nat Rev Cancer 2: 594–604, 2002.[CrossRef][Web of Science][Medline]
  40. Wali RK, Khare S, Tretiakova M, Cohen G, Nguyen L, Hart J, Wang J, Wen M, Ramaswamy A, Joseph L, Sitrin M, Brasitus T, Bissonnette M. Ursodeoxycholic acid and F(6)-D(3) inhibit aberrant crypt proliferation in the rat azoxymethane model of colon cancer: roles of cyclin D1 and E-cadherin. Cancer Epidemiol Biomarkers Prev 11: 1653–1662, 2002.[Abstract/Free Full Text]
  41. Yu J, Zhang L. The transcriptional targets of p53 in apoptosis control. Biochem Biophys Res Commun 331: 851–858, 2005.[CrossRef][Web of Science][Medline]




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