Abstract
Variations in the expression of human pregnane X receptor (hPXR)–mediated cytochrome p450 3A4 (CYP3A4) in liver can alter therapeutic response to a variety of drugs and may lead to potential adverse drug interactions. We sought to determine whether Mg2+/Mn2+-dependent phosphatase 1A (PPM1A) regulates hPXR-mediated CYP3A4 expression. PPM1A was found to be coimmunoprecipitated with hPXR. Genetic or pharmacologic activation of PPM1A led to a significant increase in hPXR transactivation of CYP3A4 promoter activity. In contrast, knockdown of endogenous PPM1A not only attenuated hPXR transactivation, but also increased proliferation of HepG2 human liver carcinoma cells, suggesting that PPM1A expression levels regulate hPXR, and that PPM1A expression is regulated in a proliferation-dependent manner. Indeed, PPM1A expression and hPXR transactivation were found to be significantly reduced in subconfluent HepG2 cells compared with confluent HepG2 cells, suggesting that both PPM1A expression and hPXR-mediated CYP3A4 expression may be downregulated in proliferating livers. Elevated PPM1A levels led to attenuation of hPXR inhibition by tumor necrosis factor-α and cyclin-dependent kinase-2, which are known to be upregulated and essential during liver regeneration. In mouse regenerating livers, similar to subconfluent HepG2 cells, expression of both PPM1A and the mouse PXR target gene cyp3a11 was found to be downregulated. Our results show that PPM1A can positively regulate PXR activity by counteracting PXR inhibitory signaling pathways that play a major role in liver regeneration. These results implicate a novel role for PPM1A in regulating hPXR-mediated CYP3A4 expression in hepatocytes and may explain a mechanism for CYP3A repression in regenerating livers.
Introduction
Cytochrome p450 (CYP)3A4 catalyzes the metabolism of more than 50% of clinically used drugs in humans (Guengerich, 1999; Veith et al., 2009). Therefore, altered expression of CYP3A4 can affect the therapeutic response to a broad spectrum of drugs and may lead to potential undesired drug interactions. Human pregnane X receptor (hPXR) is a master regulator of CYP3A4 expression (Xie et al., 2004), and altered hPXR activity could potentially lead to changes in CYP3A4 levels and activity.
The pregnane X receptor (PXR) target gene expression is regulated not only by endobiotics and xenobiotics (Kliewer et al., 1998; Lehmann et al., 1998; Staudinger et al., 2006; Pondugula et al., 2014), but also by cellular signaling pathways (Pondugula et al., 2009b). For instance, p53, FOXO1, and nuclear factor-κB (NF-κB) interact with and regulate PXR function (Kodama et al., 2004; Gu et al., 2006; Xie and Tian, 2006; Elias et al., 2013; Kodama and Negishi, 2013). Similarly, posttranslational modifications, including phosphorylation, ubiquitination, acetylation, and sumoylation, regulate PXR activity (Staudinger et al., 2011; Sugatani et al., 2012, 2014; Sivertsson et al., 2013; Smutny et al., 2013). In particular, kinases such as protein kinase A, protein kinase C, cyclin-dependent kinase 2 (CDK2), p70 S6 kinase, and Ca2+/calmodulin-dependent protein kinase II were found to modulate the activity of PXR (Staudinger et al., 2011; Elias et al., 2014; Sugatani et al., 2014). Although much is known about hPXR regulation by kinases, very little is known regarding phosphatases. We sought to determine whether Mg2+/Mn2+-dependent phosphatase 1A (PPM1A) regulates PXR activity.
PPM1A belongs to the family of metal ion-dependent Ser/Thr protein phosphatases (PPM). Phosphatases of PPM family are distinguished from other phosphatase families by their monomeric property, dependency on Mg2+/Mn2+ for activity, and insensitivity to inhibition by okadaic acid or microcystin (Lammers and Lavi, 2007; Shi, 2009). Although the PPM family currently contains 22 phosphatases, PPM1A is the most extensively characterized member of this family (Lammers and Lavi, 2007; Shi, 2009). Notably, PPM phosphatases, including PPM1A, have been recognized to regulate cell growth (Klumpp et al., 2006; Tamura et al., 2006; Lammers and Lavi, 2007; Lammers et al., 2007; Yang et al., 2011; Shohat et al., 2012). Signal cross-talk between Ser/Thr protein phosphatases and nuclear receptors has been reported. For example, PP1 and PP2A physically associate with the vitamin D receptor (Bettoun et al., 2002), and PPM1B selectively modulates peroxisome proliferator-activated receptor (PPARγ) activity (Tasdelen et al., 2013). Similarly, PPM1A interacts with vitamin D receptor (Inoue et al., 2012), which belongs to the same subfamily of nuclear receptors as PXR does. In the current study, we show that PPM1A interacts with and regulates the function of PXR.
Hepatic CYP3A levels are altered in a variety of liver pathophysiologies, including liver regeneration triggered by liver injury. CYP3A levels are significantly reduced during liver regeneration (von der Decken and Hultin, 1960; Marie et al., 1988; Habib et al., 1994; George et al., 1995; Favre et al., 1998). However, the molecular mechanisms of CYP3A repression are not fully understood. We show that PPM1A positively regulates hPXR-mediated CYP3A4 expression, and that PPM1A expression and PXR activity are regulated in a proliferation-dependent manner in HepG2 cells. We also show that the levels of both PPM1A and Cyp3a11 are downregulated in mouse regenerating livers after partial hepatectomy, and this positive correlation between PPM1A and cyp3a11 expression provides in vivo biologic relevance of PPM1A regulation of PXR-mediated CYP3A expression. Our results may explain a mechanism for CYP3A repression in regenerating livers of human patients that undergo therapeutic partial hepatectomy.
Materials and Methods
Chemicals and Plasmids.
Dimethylsulfoxide (DMSO), rifampicin, C6-ceramide, dihydro-C6-ceramide, and tumor necrosis factor α (TNFα) were purchased from Sigma-Aldrich (St. Louis, MO). The pcDNA3, FLAG-pcDNA3, pcDNA3-hPXR, FLAG-pcDNA3-hPXR, pcDNA3-PPM1A, CDK2, cyclin E, pGL3-CYP3A4-luc, and pGL3-CMV-Renilla plasmids were previously described (Lin et al., 2008; Pondugula et al., 2010, 2014; Shohat et al., 2012).
Cell Cultures, Transient Transfections, Lentiviral Short Hairpin RNA Transductions.
HepG2 human liver carcinoma cells and COS-7 monkey kidney fibroblasts were obtained from the American Type Culture Collection (Manassas, VA) and grown in Dulbecco’s modified Eagle’s medium (Lonza, Allendale, NJ) supplemented with 10% fetal bovine serum (HyClone, Logan, UT) (Pondugula et al., 2009a; Pondugula et al., 2014). HepG2 cells stably expressing hPXR and CYP3A4-luc (Lin et al., 2008) were maintained under the selection of G418. Fifty to 70% confluent HepG2 or COS-7 cells were used for most of the studies. However, we also used HepG2 cells 24 hours after reaching 100% confluency (defined as confluent HepG2 cells) as well as 30–40% confluent HepG2 cells (defined as subconfluent HepG2 cells). Transient transfections were performed using FuGENE 6 (Promega, Madison, WI) in 6-well culture plates or 55-cm2 petri dishes. HepG2 cells stably expressing hPXR and CYP3A4-luc were transduced with the lentiviral particles carrying nonsilencing control short hairpin RNA (shRNA) or PPM1A shRNA (Santa Cruz Biotechnology, Santa Cruz, CA).
PXR Transactivation Assays.
The cells were transiently transfected with pcDNA3 or FLAG-pcDNA3, pcDNA3-hPXR or FLAG-pcDNA3-hPXR, pGL3-CYP3A4-luc, pcDNA3-PPM1A, and pGL3-CMV-Renilla plasmids. After 24-hour transfection, the cells were seeded in 96-well plates (10,000 cells/well) in phenol red-free Dulbecco’s modified Eagle’s medium containing 5% charcoal/dextran-treated fetal bovine serum. The cells were then treated with the vehicle or drugs for an additional 20 or 24 hours before performing luciferase assays using the Dual-Glo luciferase assay system (Promega) and FLUOstar Optima microplate reader (BMG Labtech, Cary, NC). Firefly luciferase activity was normalized to either Renilla luciferase activity or number of liver cells measured by CellTiter-Glo luminescent cell viability assays (Promega) and shown as relative luminescence units.
Cell Proliferation and Cell Cycle Analyses.
The transduced and nontransduced HepG2 cells were plated in 96-well plates (5000 cells/well), and proliferation was determined after 48 hours using the CellTiter-Glo luminescent cell viability assay kit. For cell-cycle analysis, both subconfluent and confluent HepG2 cells in 6-well culture plates were trypsinized and processed for flow cytometry analysis (DeInnocentes et al., 2009). Briefly, the cells were fixed in 70% cold ethanol and incubated with propidium iodide to stain DNA and with RNase A to kill RNA, and analyzed for cell cycle profile by flow cytometry (ACCURI C6BD Biosciences, San Jose, CA) and CFlow Plus software.
Animals, Surgeries, and Tissue Harvesting.
Partial hepatectomy surgeries were performed on 2- to 3-mo-old male C57BL/6 mice, as previously described (Borude et al., 2012). Livers were collected at various time points between 0 and 14 days after partial hepatectomy (PHX) and processed as described before (Borude et al., 2012).
RNA Isolation and Quantitative Reverse-Transcription Polymerase Chain Reaction Assays.
Total RNA was extracted from the mouse liver tissue or HepG2 cells by using the RNeasy Mini Kit (Qiagen, Valencia, CA). Reverse transcription was performed with the QuantiTect Reverse Transcription Kit (Qiagen), and quantitative polymerase chain reaction was performed by using the QuantiTect SYBR Green Kit (Qiagen), iCycler iQ Real-Time PCR Detection System (Bio-Rad, Hercules, CA), and gene-specific primers of 18S rRNA (housekeeping gene), mouse PXR, and Cyp3a11 (Supplemental Table 1). Relative mRNA expression was calculated as described previously (Pondugula et al., 2010, 2014).
Western Blotting Analysis.
The mouse liver tissue, COS-7, or HepG2 total cell lysates were collected in radioimmunoprecipitation assay buffer, and equal amounts of protein samples were resolved on a SDS-PAGE gel and then transferred onto nitrocellulose membrane. Unbound sites on the membrane were blocked; incubated with anti-PPM1A (Abcam, Cambridge, MA), anti-FLAG (Sigma-Aldrich), or anti-actin (Santa Cruz) antibodies; washed with Tris-buffered saline; and finally incubated with horseradish peroxidase–conjugated secondary antibodies (Santa Cruz). The proteins were visualized using HyGLO Chemiluminescent HRP Antibody Detection Reagent (Denville Scientific, Metuchen, NJ).
Coimmunoprecipitation Assays.
COS-7 cells were cotransfected with FLAG-hPXR and PPM1A or FLAG-pcDNA. After 24-hour transfection, the cells were treated for additional 24 hours with DMSO or 10 µM rifampicin and lysed in Triton lysis buffer. Immunoprecipitation was carried out on 1 mg total protein using anti-FLAG M2 agarose beads (Sigma-Aldrich) for 2 hours at 4°C. The beads were washed, and the bound proteins were released by boiling in the sample loading buffer, followed by Western blot with the anti-PPM1A antibody. The same lysates were also Western blotted with the anti-PPM1A antibody.
Statistical Analysis.
Data were analyzed with Student's t test by using GraphPad Prism 6 software. Differences were considered significant (*) for P < 0.05.
Results and Discussion
PPM1A Coimmunoprecipitates with hPXR.
Recently, phosphatases including PPM family members such as PPM1A and PPM1B have been shown to functionally interact with nuclear receptors (Bettoun et al., 2002; Inoue et al., 2012; Tasdelen et al., 2013). We examined whether PPM1A interacts with hPXR. Indeed, transiently cotransfected PPM1A coimmunoprecipitated with FLAG-hPXR in COS-7 cells under basal (DMSO) and ligand (rifampicin)-stimulated conditions (Fig. 1A). This finding led us to hypothesize that PPM1A may regulate hPXR-mediated CYP3A4 expression.
PPM1A coimmunoprecipitates with hPXR, and activation of PPM1A enhances transactivation function of hPXR. (A) COS-7 cells were cotransfected with FLAG-hPXR and PPM1A or FLAG-pcDNA. Twenty-four hours post-transfection, the cells were treated with either DMSO or 10 µM rifampicin for 24 hours and lysed with Triton lysis buffer. The interaction of FLAG-hPXR with PPM1A was examined by immunoprecipitation with the anti-FLAG antibody, followed by Western blot analysis with anti-PPM1A antibody. The same lysates were also Western blotted with the anti-PPM1A antibody. Activation of PPM1A enhances transactivation function of hPXR. HepG2 (B, D, and E) and COS-7 (C) cells were cotransfected with hPXR, CYP3A4-luc, PPM1A, and CMV-Renilla (transfection control), and treated with DMSO, 1 or 10 μM rifampicin (RIF), 10 μM C6-ceramide ± 10 μM RIF, or 10 μM dihydro-C6-ceramide ± 10 μM RIF. Firefly and Renilla luciferase activities were measured 24 hours after the treatments, and the relative luminescence units were determined by normalizing with the Renilla luciferase control. Data represent mean ± S.D. from eight independent experiments. Statistical significance (*P < 0.05) was determined using unpaired Student’s t test. In (B) and (C), PPM1A-cotransfected samples were compared with hPXR-transfected samples in each treatment group.
Activation of PPM1A Leads to Increased hPXR-Mediated CYP3A4 Gene Expression.
Activation of PPM1A by overexpression led to a significant increase in basal and rifampicin-induced hPXR activation in HepG2 (Fig. 1B) and COS-7 (Fig. 1C) cells. Similarly, pharmacologic activation of PPM1A with C6-ceramide (Perry et al., 2012) resulted in increased activation of hPXR in HepG2 cells (Fig. 1D). In contrast, dihydro-C6-ceramide, an inactive analog of C6-ceramide (Lee et al., 1996), did not affect hPXR activity (Fig. 1E). These results suggest that activation of PPM1A increases hPXR-mediated CYP3A4 expression. C6-ceramide was also shown to inhibit protein kinase Cα (Lee et al., 1996), which suppresses hPXR activity (Ding and Staudinger, 2005). It is previously unknown whether C6-ceramide binds to and activates hPXR. Treatment with C6-ceramide alone did not induce the CYP3A4 promoter activity in the absence of exogenous PPM1A (Fig. 1D), suggesting that C6-ceramide is not an activator of PXR. However, it is possible that C6-ceramide–increased hPXR activity could be a combination of PPM1A activation and protein kinase Cα inhibition. Nevertheless, PPM1A overexpression studies confirm that genetic activation of PPM1A leads to upregulation of hPXR activity (Fig. 1, B and C).
Downregulation of PPM1A levels leads to impaired hPXR-mediated CYP3A4 gene expression.
Downregulation of endogenous PPM1A in HepG2 cells, which stably express hPXR and CYP3A4-luc (Lin et al., 2008), was achieved by transduction with lentiviral vectors carrying PPM1A shRNA (Fig. 2A). Knockdown of PPM1A significantly impaired both basal and rifampicin-induced transactivation function of hPXR (Fig. 2B). Nontargeting control shRNA did not affect either PPM1A expression or hPXR function (Fig. 2).
Downregulation of PPM1A levels impairs hPXR activity. (A) HepG2 cells stably expressing hPXR and CYP3A4-luc were transduced with lentiviral vectors carrying control nonsilencing shRNA or PPM1A shRNA. Whole-cell lysates were collected and subjected to Western blot analysis using anti-PPM1A and anti-actin antibodies (as a loading control). Data shown are from a representative experiment. (B) Knockdown of PPM1A impairs hPXR transactivation of CYP3A4 promoter activity. The transduced and nontransduced HepG2 cells were treated for 24 hours with DMSO or 10 µM rifampicin (RIF), and Firefly luciferase activity was measured and normalized with the total number of live cells. The relative luciferase activity is shown as the mean ± S.D. from six independent observations. Statistical significance (*P < 0.05) was determined using unpaired Student’s t test by comparing PPM1A shRNA samples with no shRNA samples in each treatment group.
Downregulation of PPM1A Promotes Proliferation of HepG2 Cells.
It is known that hPXR-activated CYP3A4 expression in hepatocytes is regulated in a proliferation-dependent manner (Lin et al., 2008; Sivertsson et al., 2013). It is also known that PPM family phosphatases, including PPM1A, regulate cell growth (Klumpp et al., 2006; Tamura et al., 2006; Lammers and Lavi, 2007; Lammers et al., 2007; Yang et al., 2011; Shohat et al., 2012). We therefore examined whether PPM1A levels affect proliferation of HepG2 cells. shRNA-targeting PPM1A, but not nontargeting shRNA, significantly increased proliferation of HepG2 cells. When proliferation of nontransduced HepG2 cells was set as 100%, proliferation of PPM1A-shRNA– and nontargeting-shRNA–transduced cells was 136% ± 8 and 103% ± 6 (P < 0.05), respectively. These results demonstrate that downregulating PPM1A expression in HepG2 cells impairs hPXR activity, but promotes proliferation.
PPM1A Expression and hPXR Activity Are Downregulated in Subconfluent HepG2 Cells.
Because PPM1A knockdown resulted in decreased hPXR activity and increased HepG2 proliferation, we determined whether PPM1A expression and hPXR activity are affected in subconfluent HepG2 cells when compared with confluent HepG2 cells. Flow cytometry analysis was performed to determine distribution of the cells in different phases of the cell cycle. Number of cells in the S phase of the cell cycle was significantly higher in the subconfluent cells (∼17%) compared with the confluent cells (∼4%) (Fig. 3A). Both PPM1A levels and hPXR transactivation of CYP3A4 promoter activity were downregulated in subconfluent HepG2 cells compared with confluent HepG2 cells (Fig. 3, B and C). These results confirm that PPM1A levels, similar to hPXR activity (Lin et al., 2008; Sivertsson et al., 2013), are regulated in a proliferation-dependent manner, and suggest that PPM1A expression and hPXR activity may be regulated in proliferating livers. We next studied the biologic relevance of PPM1A regulation of PXR-mediated CYP3A expression in HepG2 cells and mouse regenerating livers.
PPM1A protein levels (B) and hPXR activity (C) are downregulated in subconfluent proliferating HepG2 cells. (A) Both confluent and subconfluent HepG2 cells were analyzed for cell cycle distribution using flow cytometry, as described in Materials and Methods. (B) Whole-cell lysates were collected from confluent and subconfluent HepG2 cells and subjected to Western blot analysis using anti-PPM1A and anti-actin antibodies. Data shown are from a representative experiment. (C) hPXR transactivation of CYP3A4 promoter activity was determined in confluent and subconfluent HepG2 cells. The cells were transiently cotransfected with pGL3-CYP3A4-luc, CMV-Renilla (transfection control), and pcDNA3 or pcDNA3-hPXR plasmids. After 24-hour transfection, the cells were treated with DMSO or 10 µM rifampicin for another 24 hours. Firefly and Renilla luciferase activities were measured 24 hours after the treatments using Dual-Glo luciferase assay system. CYP3A4 promoter activity was determined by normalizing the Firefly luciferase activity with the Renilla luciferase control. The results are presented as fold increase over DMSO, and the values represent the mean ± S.D. of four experiments. *P < 0.05, determined by unpaired Student’s t test.
PPM1A Counteracts Inhibition of hPXR Activity by TNFα and CDK2.
It is known that CYP3A levels are repressed in proliferating hepatocytes of regenerating livers (von der Decken and Hultin, 1960; Marie et al., 1988; Habib et al., 1994; George et al., 1995; Favre et al., 1998). The hepatocyte proliferation during liver regeneration, after partial hepatectomy, occurs in the priming and proliferative phases (Michalopoulos, 2007). The priming is induced by cytokines such as TNFα primarily through NF-κB activation (Fausto et al., 1995; Galun and Axelrod, 2002), whereas the proliferation is induced by growth factors through activation of various signaling pathways, including CDK2 (Fausto et al., 1995; Gomez-Lechon et al., 1996; Galun and Axelrod, 2002; Jackson et al., 2008).
TNFα (Gu et al., 2006; Zhou et al., 2006) and CDK2 (Lin et al., 2008; Sivertsson et al., 2013; Elias et al., 2014) signaling pathways, which are upregulated and essential for hepatocyte proliferation in regenerating livers, inhibit PXR-activated CYP3A expression. Whereas CDK2 phosphorylates and inhibits PXR activity (Lin et al., 2008; Elias et al., 2014), TNFα induces NF-κB activation to inhibit PXR-retinoid X receptor (RXRα) interaction (Gu et al., 2006; Xie and Tian, 2006). As expected, TNFα (Fig. 4A) or CDK2 (overexpression of CDK2 and cyclin E) (Fig. 4B) inhibited basal and rifampicin-induced activation of hPXR.
PPM1A activation attenuates hPXR inhibition by TNFα (A) and CDK2 (B), and Cyp3a11 (C) levels correlate positively with PPM1A (D) levels in mouse regenerating livers. HepG2 cells were cotransfected with FLAG-hPXR, CYP3A4-luc, PPM1A, CDK2 (and cyclin E; data not shown), and CMV-Renilla. The cells were treated with DMSO, 10 μM rifampicin (RIF), or 20 ng/ml TNFα ± 10 µM RIF. The luciferase activities were measured 20 hours after the treatments. The relative luminescence units were shown as the means ± S.D. of five to six experiments. *P < 0.05, determined by unpaired Student’s t test. Cyp3a11 levels correlate positively with PPM1A levels in mouse regenerating livers. mRNA levels of Cyp3a11 (C) and mPXR (E) and protein levels of PPM1A (D) were shown in mouse regenerating livers at different time points (0 hours to 14 days) after PHX. mRNA levels were determined by using quantitative reverse-transcription polymerase chain reaction and by normalizing to 18S mRNA level. Protein levels were determined using Western blot analysis and by normalizing to actin protein level. Data represent mean ± S.D. (n = 3). *P < 0.05; compared with control (0 hours) by unpaired Student’s t test. Proposed mechanism for PPM1A regulation of PXR-mediated CYP3A expression in liver. (F) The current model of PXR activation via PPM1A directly or indirectly by desensitizing PXR inhibitory signaling pathways that are essential for liver regeneration. (G) One of the proposed mechanisms of CYP3A repression during liver regeneration.
PPM1A inhibits TNFα signaling by dephosphorylating and inactivating inhibitor of κB kinase (IKKβ), a central intermediate signaling molecule in TNFα-mediated activation of NF-κB (Sun et al., 2009). PPM1A also inhibits CDK2 signaling by directly dephosphorylating and inactivating CDK2 (Cheng et al., 1999, 2000). We therefore determined whether PPM1A activation attenuates TNFα and CDK2 inhibition of hPXR. Indeed, elevated PPM1A levels attenuated inhibition of hPXR by TNFα and CDK2 (Fig. 4, A and B), suggesting that PPM1A can safeguard the function of hPXR in hepatocytes by inhibiting or desensitizing TNFα and CDK2 signaling. It is possible that PPM1A attenuates TNFα inhibition of hPXR by desensitizing IKKβ phosphorylation, although we have not tested this scenario. Likewise, it remains to be determined whether PPM1A attenuates CDK2 inhibition of hPXR by dephosphorylating CDK2 and/or by dephosphorylating CDK2-phosphorylated hPXR or coregulators involved in hPXR signaling.
Cyp3a11 Expression Positively Correlates with PPM1A Expression in Mouse Regenerating Livers.
It is known that Cyp3a11 (the mouse analog of human CYP3A4) is repressed in proliferating hepatocytes of mouse regenerating livers (von der Decken and Hultin, 1960; Marie et al., 1988; Habib et al., 1994; George et al., 1995; Favre et al., 1998). However, the underlying mechanisms are not well-defined. Although PPM1A counteracts TNFα- and CDK2-mediated inhibition of PXR, these two signaling pathways are required for hepatocyte proliferation during liver regeneration. If PPM1A expression is not downregulated in proliferating hepatocytes of regenerating livers, regeneration may be affected as a consequence of PPM1A-mediated desensitization of TNFα and CDK2 signaling. It is therefore possible that PPM1A levels are downregulated in hepatocytes during liver regeneration, contributing to Cyp3a11 repression. We examined the expression of Cyp3a11, PPM1A, and mPXR in 0 hour to 14 day regenerating livers of mice after PHX (Borude et al., 2012). As expected, Cyp3a11, a mPXR target gene, was significantly downregulated in the regenerating livers (0 hours to 7 days after PHX) (Fig. 4C). Interestingly, PPM1A protein expression was also downregulated in mouse regenerating livers (0 hours to 5 days after PHX) (Fig. 4D). mPXR mRNA levels were either unaltered or higher after PHX (Fig. 4E), indicating that Cyp3a11 repression in the regenerating livers was not because of reduced mPXR levels. The positive correlation between PPM1A and Cyp3a11 expression in the regenerating livers is consistent with downregulation of PPM1A expression and hPXR-mediated CYP3A4 promoter activity in the subconfluent HepG2 cells (Fig. 3, B and C). The mouse liver regeneration studies provide in vivo biologic relevance for PPM1A regulation of PXR-mediated CYP3A expression in hepatocytes.
Although the expression of PPM1A positively correlates with CYP3A in the regenerating livers, these changes may be independent effects as partial hepatectomy induces tremendous changes in liver signaling. Further studies using PPM1A knockout mouse model would help demonstrate these associations. Similarly, whereas HepG2 cells are used for PXR characterization, primary hepatocytes are superior systems to study the regulation of drug-metabolizing enzymes. It is important to note that HepG2 cells are different from primary hepatocytes, with regard to PXR expression and signaling pathways involved in proliferation. Therefore, it would be interesting to study PPM1A regulation of PXR-mediated CYP3A4 induction in primary hepatocytes.
In summary, as shown in the proposed model, TNFα and CDK2 signaling pathways, which are vital components of liver regeneration, inhibit PXR activity (Fig. 4F). PPM1A enhances PXR-mediated CYP3A expression by counteracting the inhibitory effect of TNFα and CDK2 signaling (Fig. 4F). PPM1A and CYP3A levels are downregulated in regenerating livers compared with normal livers (Fig. 4G). It is our current proposition that decreased levels of PPM1A in regenerating livers may decrease PXR-mediated CYP3A induction and may in part contribute to repressed CYP3A levels in regenerating livers (Fig. 4G). Therefore, our results may provide a mechanism for repression of CYP3A in proliferating hepatocytes of regenerating livers of human patients, such as hepatocellular carcinoma patients, undergoing therapeutic partial hepatectomy.
Acknowledgments
The authors thank Dr. Sara Lavi (Tel Aviv University, Tel Aviv, Israel) for sharing the PPM1A plasmid and Dr. Wen Xie (University of Pittsburgh, Pittsburgh, PA) for comments. The authors also thank Drs. Coleman, Tao, Mansour, and Judd for sharing facilities and equipment.
Authorship Contributions
Participated in research design: Pondugula.
Conducted experiments: Pondugula, Flannery, Abbott, Apte, Geetha, Rege.
Contributed new reagents or analytic tools: Pondugula, Apte, Chen, Babu.
Performed data analysis: Pondugula, Flannery, Abbott.
Wrote or contributed to the writing of the manuscript: Pondugula.
Footnotes
- Received November 13, 2014.
- Accepted January 5, 2015.
This work was supported by the Animal Health and Disease Research Grant, Auburn University Intramural Grant, and Auburn University Startup Funds (to S.R.P.).
Part of this work was presented as a poster at the Nuclear Receptors and Disease meeting; 2012 Oct 30–Nov 3; Cold Spring Harbor Laboratory, New York Pondugula S.
↵
This article has supplemental material available at dmd.aspetjournals.org.
Abbreviations
- CDK2
- cyclin-dependent kinase 2
- CYP
- cytochrome p450
- DMSO
- dimethylsulfoxide
- hPXR
- human pregnane X receptor
- NF-κB
- nuclear factor-κB
- PHX
- partial hepatectomy
- PPM
- metal ion-dependent Ser/Thr protein phosphatase
- PPM1A
- Mg2+/Mn2+-dependent phosphatase 1A
- PXR
- pregnane X receptor
- shRNA
- short hairpin RNA
- TNFα
- tumor necrosis factor α
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
References
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PPM1A Regulates PXR Activity
