Tài liệu Báo cáo khoa học: Hypoxia downregulates farnesoid X receptor via a hypoxia-inducible factor-independent but p38 mitogen-activated protein kinase-dependent pathway doc

Hypoxia downregulates farnesoid X receptor via a hypoxia-inducible factor-independent but p38 mitogen-activated protein kinase-dependent pathway Tomofumi Fujino1, Kaori Murakami1, Issei Ozawa1, Yoshie Minegishi1, Ryo Kashimura1, Toshihiro Akita1, Susumu Saitou1, Takehisa Atsumi1, Takashi Sato1, Ken Ando1, Shuntaro Hara2, Kiyomi Kikugawa1 and Makio Hayakawa1 School of Pharmacy, Tokyo University of Pharmacy and Life Science, Japan School of Pharmaceutical Sciences, Showa University, Tokyo, Japan Keywords cholestasis; farnesoid X receptor; hypoxiainducible factor; ischemia; mitogen-activated protein kinase Correspondence M Hayakawa, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan Fax: +81 42 676 4508 Tel: +81 42 676 4513 E-mail: hayakawa@ps.toyaku.ac.jp (Received 18 September 2008, revised 28 November 2008, accepted 19 December 2008) doi:10.1111/j.1742-4658.2009.06867.x Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily, has been shown to play pivotal roles in bile acid homeostasis by regulating the biosynthesis, conjugation, secretion and absorption of bile acids Accumulating data suggest that FXR signaling is involved in the pathogenesis of liver and metabolic disorders Here we show that FXR expression is significantly suppressed in HepG2 cells exposed to hypoxia Concomitantly, the expression of the bile salt export pump, known as an FXR target gene product and responsible for the excretion of bile acids from the liver, is also decreased under hypoxia Overexpression of hypoxia-inducible factor (HIF)-1a does not mimic the suppressive effect of hypoxia on FXR expression Furthermore, simultaneous knockdown of HIF-1a, HIF-2a and HIF-3a fails to restore the FXR expression level under hypoxia, indicating that HIF is not involved in hypoxia-evoked FXR downregulation Instead, we demonstrate that p38 mitogen-activated protein kinase is an indispensable factor for FXR downregulation under hypoxia Thus, we propose a novel liver disorder model in which two signaling molecules, p38 mitogenactivated protein kinase and FXR, may contribute to the linkage of two pathogenic conditions, i.e ischemia, a condition accompanying hypoxia, and cholestasis, a condition with intrahepatic accumulation of cytotoxic bile acids Nuclear receptors (NRs) are ligand-activated transcriptional factors that belong to a large superfamily consisting of over 150 different members [1] Farnesoid X receptor (FXR), a member of the NR superfamily, was first isolated from a rat liver cDNA library, and named after its weak activation by supraphysiological concentrations of farnesol, an intermediate in the mevalonate biosynthetic pathway [2] Subsequent studies have revealed that certain types of bile acids act as physiological ligands for FXR, leading to its activation [3–5] FXR expression is limited to very few tissues; it is highly expressed in the liver, intestine, kidney, and adrenal gland [2,6–8], whereas lower levels of expression are reported in adipose tissues and heart [9–11] FXR forms a heterodimer with retinoid X receptor Abbreviations BSEP, bile salt export pump; CDCA, chenodeoxycholic acid; ERK, extracellular signal-regulated kinase; FXR, farnesoid X receptor; FXRE, farnesoid X receptor response element; GLUT-1, glucose transporter-1; HCC, hepatocellular carcinoma; HIF, hypoxia-inducible factor; IL, interleukin; JNK, c-Jun N-terminal kinase; LRH-1, liver receptor homolog 1; MAPK, mitogen-activated protein kinase; MRP, multidrug resistant-associated protein; NF-jB, nuclear factor kappaB; NR, nuclear receptor; SD, standard deviation; SHP, small heterodimer partner; siRNA, small interfering RNA; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS 1319 Hypoxia downregulates FXR via p38 MAPK T Fujino et al and binds to specific response elements (FXREs) on the promoters of its targeted genes [12,13] Upon ligand binding, FXR undergoes conformational changes to release corepressors and recruit coactivators, resulting in the induction of target gene expression [12,13] Activation of hepatic FXR in response to bile acids increases the expression of the FXR target gene, small heterodimer partner (SHP) SHP in turn binds to and inactivates liver receptor homolog (LRH-1), a transcription factor that is critical for CYP7A1 expression Thus, FXR activation leads to the repression of CYP7A1 transcription in an indirect manner [13] Activated FXR also increases the transcription of three hepatic transporters that function to export bile acids out of hepatocytes; the three proteins bile salt export pump (BSEP), multidrug resistant-associated protein (MRP)2 (ABCC2) and multidrug resistance P-glycoprotein (MDR3, ABCB4) are localized on the membrane of hepatocytes, and secrete bile acids from the hepatocytes into the bile canaliculi [13] Bile, containing bile acids, phospholipids, cholesterol, and proteins, is then stored in the gall bladder Bile acids excreted into bile undergo intensive enterohepatic circulation They are reabsorbed in the intestine, taken up again by the liver, and re-excreted into the bile, with this cycle being repeated several times before they are eliminated with the feces [14] In enterocytes, FXR has been shown to regulate apical sodium-dependent bile acid transporter and ileum bile acid-binding protein [13] Thus, it is clear that FXR is a key sensor for bile acids and has a central role in maintaining bile acid homeostasis Bile acid homeostasis is the result of a balance between bile acid uptake, efflux, and biosynthesis Maintenance of this balance is essential, as most bile acids are cytotoxic Cholestasis, a medical condition characterized by the impairment of normal bile flow, results in intrahepatic accumulation of cytotoxic bile acids, which cause liver injury, ultimately leading to biliary fibrosis and cirrhosis [14] As the expression of transporters responsible for bile acid export at the canalicular membrane are regulated by FXR, FXR has been thought to be a possible pharmaceutical target for the treatment of cholestasis [13] In different types of clinical situations, liver ischemia may occur and cause or contribute to hepatobiliary dysfunction, which is most often of the cholestatic type [15,16] Several lines of evidence demonstrate that the development of biliary cirrhosis is associated with the occurrence of hepatocellular hypoxia and the induction of hepatic angiogenesis [17–19] Low levels of O2 (hypoxia) are encountered by cells within rapidly growing tissues, such as developing embryos or solid 1320 tumors Most vertebrates respond to this hypoxic stress by activating the expression of a large number of genes involved in glycolysis, angiogenesis, and hematopoiesis [20] This hypoxic transcriptional response is mediated primarily by hypoxia-inducible factor (HIF), a key transcriptional regulator composed of an oxygen-regulated HIF-a subunit and the ubiquitous HIF-b (also called the aryl hydrocarbon receptor nuclear translocator) partner protein [20] HIF-a protein turnover in normoxia is very rapid, owing to the inhibitory action of the HIF-a prolyl hydroxylases These oxygen-dependent enzymes hydroxylate two conserved prolyl residues within a central oxygen-dependent degradation domain of the HIF-a proteins, promoting binding of the von Hippel–Lindau tumor suppressor protein, which acts as the ubiquitin ligase for HIF-a proteins, leading to the degradation by the proteasome [20] Any HIF-a escaping this normoxic degradation is also subjected to hydroxylation of a conserved asparagine residue within the C-terminal transactivating domain, which represses activity via abrogation of CREB-binding protein (CBP) ⁄ p300 coactivator recruitment [21] There are three HIF-a isoforms, i.e HIF-1a, HIF-2a, and HIF-3a HIF-2a is very similar to HIF-1a in both structure and function, but exhibits more restricted tissue-specific expression, and may also be differentially regulated by nuclear translocation [20] HIF-3a also exhibits conservation with HIF-1a and HIF-2a in the oxygen-dependent degradation domain; however, in contrast to HIF-1a and HIF-2a, HIF-3a does not possess a hypoxia-inducible transactivation domain, instead, having a novel C-terminus with additional uncharacterized transactivation properties [22] Several researchers have already reported that hepatocellular hypoxia causes liver angiogenesis and fibrogenesis through the inducible expression of vascular endothelial growth factor (VEGF), one of the most representative HIF target gene products [17–19] In a recent report, Fouassier et al demonstrated that the expression of BSEP and FXR was impaired in the ischemic rat liver or cultured hepatocytes exposed to hypoxia, whereas VEGF expression was elevated under the same conditions [23] However, the molecular mechanism by which hepatocellular hypoxia caused the reduced expression of the FXR and BSEP genes has not been elucidated yet Bile acids are now recognized as important regulatory molecules, not only for their own synthesis, but also for cholesterol synthesis, gluconeogenesis, glycogenesis, and apoptosis [24–26] Among various signaling pathways, mitogen-activated protein kinases (MAPKs) play important roles in transducing or modulating the bile acid-regulated cellular responses FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS T Fujino et al In mammals, four distinct subgroups of MAPKs have been identified [27] These include: (a) extracellular signal-regulated kinases (ERKs); (b) c-Jun N-terminal kinases (JNKs); (c) p38 group MAPKs; and (d) ERK5 ⁄ big MAP kinase [27] JNKs act as negative regulators of bile acid synthesis by repressing CYP7A1 expression in SHP-independent ways [14] Bile acidactivated FXR induces the expression of the FGF19 gene, resulting in the suppression of CYP7A1 through a JNK-dependent pathway [28] Alternatively, JNK activation induced by protein kinase C [29] or tumor necrosis factor (TNF) [30] also leads to the suppression of CYP7A1 expression Bile acids are also known to induce oxidative stress in mitochondria, leading to the activation of ERKs through the inactivation of protein tyrosine phosphatases [31] In contrast to JNK, p38 MAPK is reported to activate bile acid synthesis through the induction of CYP7A1 expression [32] Thus, the individual MAPK family members play different roles in maintaining bile acid homeostasis, and their imbalanced activation may cause impaired bile acid metabolism, leading to cholestasis-mediated cirrhosis In this study, we have demonstrated that FXR is significantly downregulated in HepG2 cells exposed to hypoxia Interestingly, HIFs, known as the master regulators in hypoxic responses, not participate in the hypoxia-induced downregulation of FXR In contrast, p38 MAPK, which is also known to be activated in response to hypoxia [33–35], is responsible for the downregulation of FXR under hypoxic conditions Results Cholestatic disorder is known to arise from liver ischemia [15,16] As FXR, a member of the NR superfamily, plays a key role in maintaining bile acid metabolism, we first examined whether FXR activity is impaired in cultured hepatocellular cells exposed to hypoxia Whereas FXRE-driven luciferase activity was significantly increased in HepG2 cells treated with the FXR ligand chenodeoxycholic acid (CDCA) under normoxia, cells cultured under hypoxia exhibited marginal activation induced by CDCA (Fig 1A) Similar results were obtained when Huh7 cells were used instead of HepG2 cells (Fig 1B) In order to verify whether the expression level of BSEP, a target gene of FXR, is indeed lowered under hypoxia, BSEP mRNA levels in cells under normoxia or hypoxia were compared As shown in Fig 1C, CDCA-induced elevation of BSEP mRNA was clearly demonstrated under normoxia In contrast, BSEP induction by CDCA was greatly reduced under Hypoxia downregulates FXR via p38 MAPK hypoxia, indicating that hypoxia impaired the activity of FXR, resulting in the lowered expression of BSEP Under the same hypoxic conditions, the level of nuclear factor kappaB (NF-jB) activation induced by TNF was comparable to that in cells under normoxia (Fig 1D), indicating that HepG2 cells exposed to hypoxia maintained the physiological response in terms of NF-jB activation Furthermore, when viable cell numbers were measured by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay, there was no difference between hypoxia and normoxia (data not shown) These results suggest that the hypoxia-induced downregulation of FXR activity is a physiologically regulated cellular response rather than a nonspecific result reflecting lowered cell viability Next we examined whether or not FXR expression itself is lowered in cells exposed to hypoxia As shown in Fig 2A, the level of FXR protein detected by antibody against FXR was significantly decreased in cells exposed to hypoxia as compared with that in cells under normoxia, where comparable levels of actin expression were observed under hypoxia and normoxia By measurement of the level of FXR mRNA, the lowered expression was also confirmed under hypoxia (Fig 2B) Besides FXR, LRH-1 is also known to regulate BSEP expression [36] However, under the same hypoxic conditions where the expression level of FXR was significantly lowered, LRH-1 expression was not changed at all (Fig 2C), suggesting that FXR but not LRH-1 is involved in the downregulation of BSEP under hypoxia It should be noted that the expression level of SHP, another FXR target gene, was also lowered under the same hypoxic conditions (Fig 2D) The lowered FXR mRNA level may reflect two possibilities: one is the decreased stability of FXR mRNA under hypoxia, and the other is the decreased transcription of the FXR gene in response to hypoxia Therefore, we compared FXR mRNA stability under hypoxia with that under normoxia by treating HepG2 cells with actinomycin D As shown in Fig 2E, there was no difference in the kinetics of FXR mRNA degradation between hypoxia and normoxia, suggesting that hypoxia downregulates FXR by lowering its transcription As the master regulator of hypoxic responses, HIF, a heterodimeric transcriptional factor consisting of HIF-a and HIF-b subunits, plays important roles by inducing the expression of genes required for survival of cells exposed to hypoxic environments [37] To elucidate whether or not HIF is involved in the hypoxia-evoked downregulation of FXR expression, we examined the effect of ectopically overexpressed HIF-a isoforms on FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS 1321 Hypoxia downregulates FXR via p38 MAPK T Fujino et al Fig Hypoxia-dependent suppression of FXR activity (A) HepG2 cells were cotransfected with FXRE-driven luciferase reporter vector and R reniformis luciferase expression vector phRL–TK After 24 h, CDCA (100 lM) or dimethylsulfoxide (DMSO) (vehicle control) was added to the culture, and cells were further cultured under normoxia or hypoxia for 24 h Cellular FXR activities were then determined as described in Experimental procedures Results are calculated as changes relative to the FXR activity in cells cultured under normoxia in the absence of CDCA Data are shown as the mean ± standard deviation (SD) of four determinations Similar results were obtained in four separate experiments (B) Huh7 cells were were cotransfected with FXRE-driven luciferase reporter vector and R reniformis luciferase expression vector phRL–TK, and cellular FXR activities were then determined as in (A) (C) HepG2 cells were cultured under normoxia or hypoxia for 24 h in the presence of CDCA (100 lM) or dimethylsulfoxide as vehicle control The amounts of BSEP mRNA were quantified by real-time PCR as described in Experimental procedures Results are calculated as changes relative to the amount of BSEP mRNA from cells cultured under normoxia in the absence of CDCA Data are shown as the mean ± SD of four determinations Similar results were obtained in five separate experiments (D) HepG2 cells were cotransfected with · jB–Luc luciferase reporter vector and R reniformis luciferase expression vector phRL–TK After 24 h, cells were stimulated with TNF and further cultured under normoxia or hypoxia for 24 h Results are calculated as changes relative to the NF-jB activity in cells cultured under normoxia in the absence of TNF Data are shown as the mean ± SD of four determinations Similar results were obtained in three separate experiments A B C D FXR expression in HepG2 cells HIF-a consists of three isoforms, HIF-1a, HIF-2a, and HIF-3a [38] Among three isoforms, HIF-1a is ubiquitously expressed and has been suggested to play a primary role in hypoxic responses When Pro402, Pro564 and Asn803 are replaced by alanine residues, the HIF-1a mutant becomes constitutively active even under normoxia, escaping from degradation through the ubiquitin–proteasome system Indeed, we detected a significant level 1322 of constitutively active form of HIF-a (HIF-1a) CA expression in HepG2 cells under normoxia (Fig 3A, top panel) For HIF-2a and HIF-3a, overexpression was achieved by the use of wild-type cDNAs without Pro ⁄ Ala substitutions (Fig 3A, middle and bottom panels) We first examined whether or not ectopically overexpressed HIF-a isoforms indeed function in HepG2 cells under normoxia As shown in Fig 3B, HIF-1a CA and HIF-3a significantly induced glucose transporter-1 (GLUT-1), a well-known HIF target gene [39], whereas ectopically overexpressed HIF-2a failed to elevate its level As HIF-a isoforms are known to function in cell type-specific or target gene-specific ways [39], HIF-2a may not be active in terms of GLUT-1 regulation in HepG2 cells Although at least HIF-1a and HIF-3a were shown to be functional, neither of them lowered the FXR protein expression level (Fig 3C) Furthermore, when FXR mRNA levels were examined, none of the HIF-a isoforms could mimic the effect of hypoxia, whereas slight but significant increases in FXR mRNA were observed in cells overexpressing HIF-1aCA and HIF-3a (Fig 3D) To confirm that HIF is not involved in FXR downregulation under hypoxia, the expression level of FXR was examined in HepG2 cells in which endogenous HIF-a isoforms were simultaneously knocked down FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS T Fujino et al A B C D E As shown in Fig 4A, the expression levels of endogenous HIF-1a and HIF-2a were significantly elevated in cells exposed to hypoxia When cells were transfected Hypoxia downregulates FXR via p38 MAPK Fig Hypoxia suppresses the transcription of FXR (A) HepG2 cells cultured under normoxia or hypoxia for 24 h were lysed in SDS ⁄ PAGE sample buffer, and the resultant cell lysates were subjected to immunoblotting analyses to detect FXR or to detect b-actin levels as a loading control Similar results were obtained in three separate experiments (B) HepG2 cells were cultured under normoxia or hypoxia for 24 h, and total RNAs were then prepared The amounts of FXR mRNA were quantified by real-time PCR as described in Experimental procedures Results are calculated as changes relative to the amount of FXR mRNA from cells cultured under normoxia Data are shown as the mean ± SD of four determinations Similar results were obtained in three separate experiments (C) HepG2 cells were cultured under normoxia or hypoxia for 24 h, and the amounts of LRH-1 mRNA were quantified by realtime PCR as in (B) Results are calculated as changes relative to the amount of LRH-1 mRNA from cells cultured under normoxia Data are shown as the mean ± SD of four determinations Similar results were obtained in three separate experiments (D) HepG2 cells were cultured under normoxia or hypoxia for 24 h, and the amounts of SHP mRNA were quantified by real-time PCR as in (B) Results are calculated as changes relative to the amount of SHP mRNA from cells cultured under normoxia Data are shown as the mean ± SD of four determinations Similar results were obtained in three separate experiments (E) HepG2 cells were treated with actinomycin D (5 lM) and then cultured under normoxia or hypoxia for indicated times The amounts of FXR mRNA were quantified by real-time PCR as described in Experimental procedures Results are calculated as changes relative to the amount of FXR mRNA at h Data are shown as the mean ± SD of four determinations Similar results were obtained in three separate experiments with the combined mixture of small interfering RNAs (siRNAs) against HIF-1a, HIF-2a, and HIF-3a, the levels of HIF-1a and HIF-2a in cells under hypoxia were drastically decreased (Fig 4A) In the case of HIF-3a, endogenous HIF-3a was not significantly elevated under hypoxia; therefore, knockdown efficiency was evaluated using HepG2 cells that ectopically overexpressed HIF-3a As shown in Fig 4B, the combined transfection of three siRNAs against HIF-1a, HIF-2a and HIF-3a also effectively reduced the expression level of HIF-3a However, under the condition where three isoforms of HIF-a were significantly knocked down, the lowered expression of FXR protein in cells under hypoxia was not restored at all (Fig 4C) Similarly, at the level of mRNA, hypoxia-dependent downregulation of FXR was not restored by the triple knockdown of three HIF-a isoforms (Fig 4D) These results clearly demonstrated that HIFs not participate in the downregulation of FXR in cells under hypoxia Next, we focused on MAPK families as candidates for upstream signaling molecules responsible for the downregulation of FXR in response to hypoxia, as bile acid homeostasis is regulated in many aspects by each subfamily of MAPKs [14,32] and, in addition, several FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS 1323 Hypoxia downregulates FXR via p38 MAPK T Fujino et al A A B B C D C D Fig Ectopically overexpressed HIF-a isoforms fail to suppress FXR expression (A) HepG2 cells were transfected with pcDNA3.1 as a vehicle control or with pcDNA3.1–HIF-1a CA, pcDNA3.1–HIF2a, or pcDNA3.1–HIF-3a After 24 h, cell lysates were prepared, and immunoblotting analyses to detect HIF-1a, HIF-2a and HIF-3a then performed (B) HepG2 cells were transfected with pcDNA3.1 as a vehicle control or with pcDNA3.1–HIF-1a CA, pcDNA3.1–HIF2a, or pcDNA3.1–HIF-3a After 24 h, total RNAs were prepared, and real-time PCR analysis to detect GLUT-1 mRNA was then performed as described in Experimental procedures (C) As described in (A), cell lysates were prepared, and immunoblotting analyses were then performed to detect FXR or to detect b-actin levels as a loading control (D) HepG2 cells were transfected with pcDNA3.1 as a vehicle control or with pcDNA3.1–HIF-1a CA, pcDNA3.1– HIF-2a, or pcDNA3.1–HIF-3a After 24 h, total RNAs were prepared, and real-time PCR analysis was then performed to detect FXR mRNA, as described in Experimental procedures Fig Knockdown of endogenous HIF isoforms does not restore FXR expression under hypoxia (A) HepG2 cells were transfected with control siRNA (#1022076) or with the combined mixture of siRNAs against HIF-1a, HIF-2a, and HIF-3a, and then cultured under normoxia or hypoxia for 24 h Cell lysates were then prepared, and immunoblotting analyses were then performed to detect HIF-1a and HIF-2a (B) In order to estimate the knockdown efficiency of HIF-3a, HepG2 cells transfected with pcDNA3.1–HIF-3a were then treated with the combined mixture of siRNAs against HIF-1a, HIF-2a, and HIF-3a, and then cultured under normoxia for 24 h Cell lysates were then prepared, and this was followed by immunoblotting analysis to detect HIF-3a (C) As described in (A), cell lysates were prepared, and then immunoblotting analyses were performed to detect FXR or to detect b-actin levels as a loading control (D) HepG2 cells transfected with the combined mixture of siRNAs against HIF-1a, HIF-2a and HIF-3a were then cultured under hypoxia for 24 h After 24 h, total RNAs were prepared, and then real-time PCR analysis was performed to detect FXR mRNA as described in Experimental procedures lines of evidence indicate that hypoxia induces the activation of MAPK families [33–35,40,41] The activation of each MAPK was examined by the use of antibodies that detect phosphorylated forms of MAPKs (Fig 5) Before cells were exposed to hypoxia, the phosphorylated form of p38 was retained at a low level; however, its level was significantly increased during hypoxia, becoming more than 10 times higher after h of 1324 FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS T Fujino et al Fig Activation of p38 and ERK1 ⁄ MAPKs in response to hypoxia occurs in inverse relation to the downregulation of FXR HepG2 cells were cultured under hypoxia for the indicated times Cell lysates were then prepared, and this was followed by immunoblotting analyses to detect FXR, phosphorylated forms of p38 and ERK1 ⁄ or b-actin levels as a loading control Quantification of the bands was done by densitometric analysis (IMAGE GAUGE 4.0) Similar results were obtained in three separate experiments hypoxia treatment (Fig 5) On the other hand, phosphorylation of ERK2 was observed before hypoxia treatment Hypoxia treatment for h resulted in a two-fold elevation of phospho-ERK2 level, whereas only slight activation of ERK1 was observed under hypoxia (Fig 5) During this hypoxia treatment, the expression levels of FXR were inversely lowered in a time-dependent manner (Fig 5) It should be noted that we could not detect the activation of JNK using anti-phospho-JNK antibodies in HepG2 cells exposed to hypoxia, whereas the same antibodies can detect the activation of JNK in L929 cells treated with TNF (data not shown) These results suggest that p38 or ERK1 ⁄ but not JNK may participate in the downregulation of FXR during hypoxia In many studies concerning MAPK signaling, pharmacological inhibitors specific for each subfamily of MAPKs have been widely used However, the action of these compounds must be considered with caution, as they sometimes affect other signaling pathways in unexpected ways Indeed, for unknown reasons, SB203580, which is widely used as a p38-specific inhibitor, lowered FXR expression level by itself but increased phosphorylated forms of p38 in HepG2 cells (data not shown) There are many reports indicating that SB203580 inhibits different types of kinases, such as Akt ⁄ protein kinase B [42] and RIP ⁄ RICK [43], independently of p38 inhibition Furthermore, SB203580 was shown to activate ERK in human hepatocytes and HepG2 cells [44,45] Therefore, we carried out RNA interference experiments in order to verify the role of p38 and ERK1 ⁄ 2, instead of using pharmacological inhibitors Hypoxia downregulates FXR via p38 MAPK The expression level of p38 was examined by immunoblotting using antibody against p38 followed by densitometric analysis (Fig 6A) The amount of p38 protein derived from cells treated with the p38a siRNA under hypoxia was reduced to 61% when expressed as a percentage of the value of the ‘normoxia’ control (Fig 6A, middle panel) It should be noted that the increase in the concentration of p38a siRNA did not improve the knockdown efficiency of p38, but rather resulted in impairment of cell viability (data not shown) In spite of this partial knockdown efficiency, p38a siRNA treatment sufficiently reversed the hypoxia-dependent downregulation of FXR As shown in the top panel of Fig 6A, the amount of FXR protein under hypoxia was 27% of the value of the ‘normoxia’ control, and the treatment of cells with p38a siRNA increased the FXR protein level to 42% even under hypoxia (Fig 6A, top panel) The restorative effect of p38 siRNA was also confirmed by the quantification of FXR mRNA As shown in Fig 6B, the lowered FXR mRNA level under hypoxia was significantly elevated by treatment of cells with p38a siRNA The reduced BSEP mRNA expression under hypoxia was also restored by the same treatment with p38a siRNA (Fig 6C) As a stress-activated MAPK, p38 is known to be activated by various stimuli, including proinflammatory cytokines As shown in Fig 6D, interleukin (IL)-1b induced strong p38 activation in HepG2 cells Interestingly, the FXR level was reduced by treatment with IL-1b (Fig 6D, top panel) These results suggest that p38 acts as an upstream signaling molecule that responds to various environmental stresses, including hypoxia, and downregulates FXR transcription We next examined the role of ERK1 ⁄ in hypoxiadependent downregulation of FXR As shown in Fig 6E, simultaneous knockdown of ERK1 and ERK2 reduced the ERK2 protein level to 44% of the value of the ‘normoxia’ control (Fig 6E, middle panel) ERK1 expression was also decreased in cells treated with ERK1 ⁄ siRNAs Similar to the case of p38a siRNA, higher concentrations of siRNA against ERK1 ⁄ did not provide good knockdown efficiency (data not shown) Under the condition described above, the lowered expression of FXR protein in cells exposed to hypoxia (48% of the value of the ‘normoxia’ control) was not elevated at all by the treatment with ERK1 ⁄ siRNAs (41%), as shown in the top panel of Fig 6E As we could not achieve the complete knockdown of ERK1 ⁄ 2, we could not rule out the involvement of ERK1 ⁄ in the hypoxia-dependent downregulation of FXR However, we can highlight p38 MAPK as the key molecule responsible for the FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS 1325 Hypoxia downregulates FXR via p38 MAPK T Fujino et al Fig p38 but not ERK1 ⁄ MAPK is involved in the hypoxiadependent downregulation of FXR (A) HepG2 cells were transfected with control siRNA or with the siRNA against p38a, and then cultured under hypoxia for 24 h Cell lysates were then prepared, and this was followed by immunoblotting analyses to detect FXR and p38 or b-actin levels as a loading control Quantification of the bands were done by densitometric analysis (IMAGE GAUGE 4.0) Similar results were obtained in three separate experiments (B) HepG2 cells transfected with control siRNA or with the siRNA against p38a were cultured under hypoxia for 24 h Total RNAs were then prepared, and real-time PCR analysis to detect FXR mRNA was carried out as described in Experimental procedures Similar results were obtained in three separate experiments (C) HepG2 cells transfected with control siRNA or with the siRNA against p38a were cultured under hypoxia for 24 h Total RNAs were then prepared, and real-time PCR analysis to detect BSEP mRNA was performed as described in Experimental procedures Similar results were obtained in three separate experiments (D) HepG2 cells cultured under normoxia were stimulated with ngỈmL)1 IL-1b for h Cells were then lysed in SDS ⁄ PAGE sample buffer, and the resultant cell lysates were subjected to immunoblotting analyses to detect FXR, phosphorylated forms of p38 or to detect b-actin levels as a loading control Quantification of the bands was done by densitometric analysis (IMAGE GAUGE 4.0) Similar results were obtained in three separate experiments (E) HepG2 cells were transfected with control siRNA (#1022076) or with the combined mixture of siRNAs against ERK1 and ERK2 then cultured under hypoxia for 24 h Cell lysates were then prepared followed by the immunoblotting analyses to detect FXR and ERK1 ⁄ or b-actin levels as a loading control Quantification of the bands was done by densitometric analysis (IMAGE GAUGE 4.0) Similar results were obtained in three separate experiments A B C Discussion D E negative regulation of FXR transcription, as its activation occurs more drastically than that of ERK1 ⁄ under hypoxia (Fig 5), its partial knockdown is effective enough to restore the FXR function impaired by the hypoxia treatment (Fig 6A–C), and its activation by IL-1b also leads to the downregulation of FXR (Fig 6D) 1326 In the current study, we have demonstrated that FXR, a key transcription factor that regulates bile acid metabolism, is downregulated under hypoxia through a p38 MAPK-dependent mechanism The experimental model shown here may give an explanation of how chronic ischemia impairs liver function by attenuating the bile acid homeostasis regulated by FXR and possibly leads to progressive liver disorders such as primary biliary cirrhosis and primary sclerosing cholangitis Indeed, an ischemia-induced low-oxygen condition, i.e hypoxia, has been thought of as a possible cause of bile duct injury, in particular after liver transplantation, hepatic surgery, and intra-arterial chemotherapy [23,46,47] Hypoxia is a serious stress for living organs, because of the need to make the massive change from oxygendependent to oxygen-independent energy production Therefore, the ability to sense and respond to changes in oxygen is essential for the survival of multicellular organisms HIF is the key transcription factor for sensing and responding to lowered oxygen, acting by inducing the transcription of various genes required FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS T Fujino et al for survival under conditions where the oxygen supply is limited VEGF is one such HIF-targeted gene, and is known to be upregulated in the cirrhotic liver [17] During the development of biliary cirrhosis, hepatocellular hypoxia and hepatic angiogenesis induced by VEGF are known to contribute to the progression of liver fibrosis [18] Moreover, enhanced proliferation of liver tumor cells leads to local hypoxia in hepatocellular carcinoma (HCC) [48], and in turn, hypoxiainduced expression of angiogenic factors such as VEGF results in the hypervascularity of HCC Undoubtedly, HIF and VEGF play central roles during new vessel formation in HCC [48] In contrast, our present study has revealed that HIF is not involved in hypoxia-dependent downregulation of FXR Thus, hypoxia may affect liver disorders by activating two signaling pathways: one is the HIF-dependent pathway that leads to the induction of VEGF, resulting in hypervascularity in the liver; and the other is the HIF-independent but p38-mediated pathway that causes down-egulation of FXR and BSEP, resulting in a decreased capacity for bile acid excretion from the liver through the biliary tracts However, we should not rush to the conclusion that the downregulated FXR expression under hypoxia should be primarily restored to avoid the progress of cholestatic liver disorders, as cholestasis results either from an impairment of bile secretion or from an obstruction of the bile duct [49] As suggested by Fiorucci et al., activation of canalicular transporters, including BSEP, could negatively impact on intrahepatic bile duct pressure in patients with severely obstructed bile flow [12] This concept is supported by the observation that FXR) ⁄ ) mice develop less hepatic injury in response to bile duct ligation than wild-type mice [50,51] Besides BSEP, the MRPs are known to be involved in the export of bile acids from the liver [52] Among six MRP subfamilies (MRP1–6), four (MRP1–4) are expressed in the liver [53] MRP3 and MRP4 localize in the basolateral membrane of the hepatocyte, whereas MRP2 is expressed on the canalicular membrane in a similar manner as BSEP Interestingly, bile duct obstruction in FXR) ⁄ ) mice causes robust induction of MRP4 in the basolateral membrane [50] As this effect is not observed in wild-type mice, FXR might negatively regulate the activation of basolateral efflux of bile acid from hepatocytes [50,51] Moreover, the hepatic expression of human MRP3 is usually very low; however, it is induced in patients with cholestasis and cirrhosis [54] Thus, the basolateral MRPs upregulated during severe cholestasis may act as an alternative export system to eliminate bile acids from the liver Hypoxia downregulates FXR via p38 MAPK by elevating bile acid efflux across the basolateral membrane of the hepatocyte, instead of their being excreted through the bililary tract using transporters located on the canalicular membrane Therefore, hypoxia-evoked downregulation of FXR and BSEP may have opposite effects on the progress of cholestasis, depending on whether or not bile flow is severely obstructed Under conditions where biliary tract is not yet injured, the decreased bile flow that results from the downregulation of FXR ⁄ BSEP in response to hypoxia will lead to the accumulation of toxic bile acids in hepatocytes, and cholestasis will consequently be promoted It should be noted that genetic defects of BSEP cause a severe liver disease in humans called progressive familial intrahepatic cholestasis type 2, which leads to irreversible liver damage, owing to intrahepatic bile acid accumulation [55] In addition, decreased FXR expression and activity is known to be associated with FIC1 mutations, suggesting that FXR itself may play an important role in the pathogenesis of progressive familial intrahepatic cholestasis type [56,57] As we have shown in our present study, the expression level of FXR is dynamically changed in cells in response to extracellular stimuli, such as hypoxic stress Thus, at the early stage of ischemia, when bile flow is not yet severely obstructed, hypoxia-evoked FXR ⁄ BSEP downregulation may lead directly to cholestasis In contrast, under conditions where the bile duct is severely obstructed, the use of ‘retrograde’ alternative and basolateral transporters instead of ‘orthograde’ canalicular transporters will be beneficial for the liver In this context, at the late stage of chronic ischemia, the p38 MAPK signaling pathway may constitute the molecular switch system that turns off the FXR-regulated canalicular transporters in response to hypoxia, and turns on the alternative basolateral transporters in order to minimize the intrahepatic accumulation of bile acids, although direct evidence for this has not yet been provided Interestingly, Kubitz et al reported that the treatment of HepG2 cells with cycloheximide, an inhibitor of protein translation, induced trafficking of BSEP from the Golgi to the canalicular membrane in a p38 MAPK-dependent manner [58] It is likely that p38 MAPK activated in response to hypoxia allows BSEP to be expressed in the canalicular membrane at a minimal level while it turns off the FXR-regulated transcription of BSEP Bile acid metabolism is tightly regulated via a complex network of signaling pathways Among them, FXR and MAPKs have major roles, and imbalanced outputs of their signals will lead to liver disorders There have been several reports showing crosstalk between FXR-regulated pathways and MAPK FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS 1327 Hypoxia downregulates FXR via p38 MAPK T Fujino et al signaling pathways [14,28–32] In this study, we have highlighted p38 MAPK and FXR as the signaling molecules that may play key roles in the pathogenesis of the liver disorders accompanying ischemia and cholestasis Although the molecules that act downstream of p38 MAPK to suppress the function of FXR remain to be elucidated, we believe that our present study contributes to the understanding of the molecular basis of cholestasis progressing under ischemia Further insights into the crosstalk between p38 MAPK and FXR will be useful in identifying a novel therapeutic target for this type of liver disorder firefly luciferase reporter vector pGL4–FXREx4 (0.4 lg) containing four copies of the FXRE consensus sequence (GGGTCAGTGACCC) and Renilla reniformis luciferase expression vector phRL–TK (0.04 lg) were cotransfected into cells using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions After 24 h of incubation, cells were treated with 100 lm CDCA, and then further incubated in the hypoxia workstation or left under the regular culture condition (normoxia) for 24 h Cells were then harvested, and cellular firefly and Renilla luciferase activities were measured using a chemiluminescense photometer Firefly luciferase activity was normalized with that of Renilla luciferase Data were analyzed by Student’s t-test Experimental procedures NF-jB activity assay Antibodies Antibodies specific for b-actin (C-2), FXR (D-3) and HIF-3a (H-170) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) Antibodies against p38, phospho-p38 (Thr180 ⁄ Tyr182) and phospho-ERK1 ⁄ were obtained from Cell Signalling Technology (Danvers, MA, USA) Antibody against HIF-1a (clone 54) was a product of BD Biosciences Pharmingen (Franklin Lakes, NJ, USA) Antibody against HIF-2a (EP-190b) was obtained from Novus Biologicals (Littleton, CO, USA) ECL anti-mouse IgG, horseradish peroxidase-linked whole antibody (from sheep), and ECL anti-rabbit IgG, horseradish peroxidaselinked whole antibody (from donkey), were purchased from GE Healthcare (Little Chalfont, UK) TNF-induced NF-jB activation in HepG2 cells exposed to hypoxia was assessed by dual luciferase assay (Promega) HepG2 cells were seeded on a 24-well culture plate at a density of · 105 cells per well and cultured for 24 h, and · jB–Luc luciferase reporter vector (0.4 lg) and phRL– TK (0.04 lg) were cotransfected into cells using Lipofectamine 2000 transfection reagent (Invitrogen) After 24 h of incubation, cells were treated with or 10 ngỈmL)1 TNF, and then further incubated in the hypoxia workstation or left under the regular culture condition (normoxia) for 24 h Cells were then harvested, and cellular firefly and Renilla luciferase activities were measured using a chemiluminescense photometer Firefly luciferase activity was normalized with that of Renilla luciferase Cell culture Human hepatocellular carcinoma cell lines HepG2 and Huh7 were cultured in DMEM containing 10% fetal bovine serum, 50 unitsỈmL)1 penicillin and 50 lgỈmL)1 streptomycin in a humidified atmosphere of 8.5% CO2 at 37 °C Hypoxia experiments Hypoxia (< 3%) was obtained in a workstation with O2 and CO2 and temperature control (Ikemoto Rika Co., Tokyo, Japan) For hypoxic harvesting, cells were lysed in the workstation with buffers equilibrated in the hypoxic condition Cell viability during hypoxia was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay [59] Immunoblotting Cells were washed with NaCl ⁄ Pi, and cell extracts were prepared using SDS sample buffer After normalization of protein content by the protein assay, samples were separated by SDS ⁄ PAGE and subjected to immunoblotting analysis For the detection of FXR, p38, phospho-p38, and phospho-ERK1 ⁄ 2, poly(vinylidene difluoride) membranes were incubated with the primary antibody for h at room temperature, and this was followed by incubation for 16 h at °C For the detection of ERK1 ⁄ 2, HIF-1a, HIF-2a, HIF-3a, and b-actin, the membranes were incubated with the primary antibody for h Immunocomplexes on the poly(vinylidene difluoride) membranes were visualized with enhanced chemiluminescence western blotting detection reagents (GE Healthcare Biosciences) Measurement of FXR activity Cellular FXR activity was measured by dual luciferase assay (Promega, Madison, WI, USA) HepG2 cells were seeded on a 24-well culture plate at a density of · 105 cells per well and cultured for 24 h FXRE-driven 1328 Quantification of mRNAs The amounts of mRNAs were quantified by real-time PCR Briefly, lg of total RNAs were reverse-transcribed by the use of Ready-to-Go-You-Prime First-Strand Beads (GE FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS T Fujino et al Healthcare Biosciences) according to the manufacturer’s instructions, and the resultant cDNAs were then subjected to real-time PCR analysis using a TaqMan Gene Expression Assay kit (Applied Biosystems, Foster City, CA, USA) For the detection of FXR, BSEP, LRH-1, SHP, GLUT-1 and b-actin mRNAs, TaqMan assay mixtures Hs00231968, Hs00184824, Hs00187067, Hs0022677, Hs00892681 and 4310881E were used, respectively TaqMan assay mixture Hs00231968 can detect all of the four isoforms of FXR [60] Amplification and quantification were done with the PRISM 7000 Real-Time PCR System (Applied Biosystems) FXR, BSEP, LRH-1, SHP and GLUT-1 mRNA levels were normalized to the levels of b-actin mRNA as an internal control Data were analyzed by Student’s t-test Construction of the constitutively active HIF-1a expression vector We have previously used the expression vector encoding the mutant of HIF-1a, in which Pro564 and Asn803 are replaced by alanine [61], as this mutant was expected to act as a constitutively active version of HIF-1a [21] In addition to Pro564, Pro402 in HIF-1a is now recognized as the target residue of prolyl hydroxylase; therefore, we made an additional mutation, replacing Pro402 with the alanine residue In brief, we performed site-directed mutagenesis by PCR as follows The pBluescript SK vector (Stratagene, La Jolla, CA, USA) encompassing an HIF-1a cDNA in which Pro564 and Asn808 were replaced with alanines was used as a template Two sets of primers [set 1, 5¢-TAGTCCAG TGTGGTGGAATTCTGC-3¢ (sense) and 5¢-AAAGCATC AGGTTCCTTCTTAAG-3¢ (antisense); set 2, 5¢-AACTTT GCTGGCCGCCGCCGCTGG-3¢ (sense) and 5¢-GGCAAC TAGAAGGCACAGTCGAGG-3¢ (anti-sense)] were used to generate the substitution of Pro402 for the alanine residue The resultant cDNA was subcloned into pcDNA3.1 vector (Invitrogen) and termed pcDNA3.1–HIF-1a CA Hypoxia downregulates FXR via p38 MAPK According to the report by Sowter et al [62], custom siRNAs against HIF-1a and HIF-2a were prepared as follows The mixture of the sense (5¢-CUGAUGACCA GCAACUUGAdTdT-3¢) and antisense (5¢-UCAAGUUGC UGGUCAUCAGdTdT-3¢) oligonucleotides was denatured at 90 °C, cooled for annealing, and used to knock down HIF-1a Similarly, the siRNA against HIF-2a was prepared using the sense (5¢-CAGCAUCUUUGAUAGCAGUdTdT3¢) and antisense (5¢-ACUGCUAUCAAAGAUGCUGdT dT-3¢) oligonucleotides To knock down HIF-3a, HP siRNA (Hs_HIF3A_1_HP siRNA) was used In order to knock down endogenous HIF-1a, HIF-2a and HIF-3a simultaneously, HepG2 cells were seeded on 35 mm dishes at a density of · 105 cells per dish and cultured for 24 h, and this was followed by transfection with the combined mixture of siRNAs against HIF-1a, HIF2-a, and HIF3a (150 nm each), using Oligofectamine Reagent (Invitrogen) according to the manufacturer’s instructions On the day after the first transfection, a second transfection was performed similarly to the first [63] Cells were then cultured under normoxic or hypoxic conditions for 24 h, and protein extracts or total RNAs were prepared for immunoblotting or real-time PCR analyses To knock down endogenous p38a or ERK1 ⁄ expression, we used the following validated siRNAs: Hs_MAPK14_6_HP validated siRNA against human p38a; Hs_MAPK3_7_HP validated siRNA against human ERK1; and Hs_MAPK1_10_HP validated siRNA against human ERK2 HepG2 cells were seeded on 35 mm dishes at a density of · 105 cells per dish Immediately after seeding, cells were transfected with p38a siRNA (10 nm), or with the mixture of ERK1 siRNA and ERK2 siRNA (10 nm each), using HiPerfect Transfection Reagent (Qiagen) according to the manufacturer’s instructions Cells were then cultured under hypoxic conditions for 24 h, and protein extracts or total RNAs were prepared for immunoblotting or real-time PCR analyses In the series of RNA interference experiments, ‘nonsilencing control’ siRNA (#1022076) from Qiagen was used as a control Transfection of HIF-a isoform HepG2 cells were seeded on 35 mm dishes at a density of · 105 cells per dish and cultured for 24 h Cells were then transfected with pcDNA3.1–HIF-1a CA, pcDNA3.1–HIF2a or pcDNA3.1–HIF-3a by the use of Lipofectamine 2000 transfection reagent (Invitrogen) After 24 h, protein extracts or total RNAs were 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[set 1, 5¢-TAGTCCAG TGTGGTGGAATTCTGC-3¢ (sense) and 5¢-AAAGCATC AGGTTCCTTCTTAAG-3¢ (antisense); set 2, 5¢-AACTTT GCTGGCCGCCGCCGCTGG-3¢ (sense) and 5¢-GGCAAC TAGAAGGCACAGTCGAGG-3¢ (anti-sense)]