AJP - GI Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 293: G809-G816, 2007. First published August 2, 2007; doi:10.1152/ajpgi.00212.2007
0193-1857/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/4/G809    most recent
00212.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Radaeva, S.
Right arrow Articles by Gao, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Radaeva, S.
Right arrow Articles by Gao, B.

LIVER AND BILIARY TRACT

Retinoic acid signaling sensitizes hepatic stellate cells to NK cell killing via upregulation of NK cell activating ligand RAE1

Svetlana Radaeva,1 Lei Wang,1 Sergei Radaev,2 Won-Il Jeong,1 Ogyi Park,1 and Bin Gao1

1Section on Liver Biology, Laboratory of Physiologic Studies, National Institute on Alcohol Abuse and Alcoholism and 2Structural Immunology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland

Submitted 10 May 2007 ; accepted in final form 19 July 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hepatic stellate cells (HSCs) store 75% of the body's supply of vitamin A (retinol) and play a key role in liver fibrogenesis. During liver injury, HSCs become activated and susceptible to natural killer (NK) cell killing due to increased expression of the NK cell activating ligand retinoic acid early inducible gene 1 (RAE-1). To study the mechanism by which RAE-1 is upregulated in HSCs during activation, an in vitro model of cultured mouse HSCs was employed. RAE-1 was detected at low levels in quiescent HSCs but upregulated in 4- and 7-day cultured HSCs (early activated HSCs), whereas 21-day cultured HSCs (fully activated HSCs) lost RAE-1 expression. High levels of RAE-1 in 4- and 7-day cultured HSCs correlated with their susceptibility to NK cell killing, which was diminished by treatment with RAE-1 neutralizing antibody. Furthermore, retinoic acid (RA) and retinal dehydrogenase (Raldh) levels were upregulated in early activated HSCs compared with quiescent or fully activated HSCs. Blocking RA synthesis by the Raldh inhibitor or blocking RA signaling by the retinoic acid receptor antagonist abolished upregulation of RAE-1 whereas treatment with RA induced RAE-1 expression in HSCs. In conclusion, during activation, HSCs lose retinol, which is either secreted out or oxidized into RA; the latter stimulates RAE-1 expression and sensitizes early activated HSCs to NK cell killing. In contrast, fully activated HSCs become resistant to NK cell killing because of lack of RAE1 expression, leading to chronic liver fibrosis and disease.

retinoic acid; stellate cells; NK cells; RAE-1; liver fibrosis

UNDER NORMAL HEALTHY CONDITIONS, the hepatic stellate cells (HSCs) are quiescent and store 75% of the body's supply of retinol; however, in response to liver injury of any etiology, the quiescent HSCs are converted into myofibroblast-like cells that are highly proliferative, fibrogenic, and contractile. These activated HSCs play an essential role in the development and progression of liver fibrosis (3, 13, 14, 17, 28, 31). In vitro, HSCs cultured on plastic dishes can be spontaneously activated, displaying a myofibroblastic phenotype that is fundamentally similar to HSC activation in vivo (3, 13, 14, 17, 28, 31). HSC activation can be divided into early and chronic stages of activation, which are distinguishable according to cell morphology and gene expression. HSCs cultured for 4–7 days become characteristically early activated HSCs, which lose their stores of retinol through either secretion or dehydrogenation into retinal by alcohol dehydrogenase (ADH) and conversion into retinoic acid (RA) by retinal dehydrogenase (Raldh) (5, 8, 9, 14, 32). In contrast, cells cultured for long periods of time (21 days) act like fully activated HSCs with myofibroblast-like functionality (3, 13, 14, 17, 28, 31). Activated HSCs produce collagens, leading to liver fibrosis (3, 13, 14, 17, 28, 31).

Recent evidence suggests that liver fibrosis and even cirrhosis can be reversed, with clearance of HSCs through apoptosis as a key step in this process (10–12, 29). Although factors involved in HSC apoptosis during liver fibrosis are not fully understood, we and others have recently demonstrated that activated HSCs during liver fibrosis become susceptible to natural killer (NK) cell killing, which may be an important mechanism contributing to HSC apoptosis (20, 21, 30). The sensitivity of activated HSCs to NK cell killing is likely mediated by two related processes (21, 30): induction of retinoic acid-induced early gene (RAE-1), an NK cell activating ligand, and downregulation of NK cell inhibitory ligand (major histocompatibility complex I). The signaling that controls both these processes is unclear at this time. The RAE-1 protein was originally isolated from mouse embryonic carcinoma F9 cells as RA-inducible clones (24) and later identified as an NKG2D ligand that activates NK cells (6). Although expression of RAE-1 mRNA is detectable during embryogenesis, it is not typically expressed in adult tissue (24). High levels of RAE-1 was also detected in the liver of hepatitis B virus transgenic mice and the dorsal root ganglia neurons; however, the underlying mechanisms remain unknown (2, 7). Recently, we have demonstrated that high levels of RAE-1 mRNA were detected in activated HSCs but not in quiescent HSCs from C57Bl/6 mice (30). Hence, the mechanisms underlying RAE-1 upregulation in activated HSCs are investigated in this paper by using an in vitro culture model. Our findings suggest that activation of RA signaling plays an important role in induction of RAE1 expression in early activated HSCs and subsequently sensitizes these cells to NK cell killing.


   METHODS AND MATERIALS
TOP
 ABSTRACT
 METHODS AND MATERIALS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice. C57Bl/6N mice were purchased from the National Cancer Institute (Frederick, MD). All mice were used in accordance with institutional guidelines for animal experimentation. The protocol for this study was approved by the National Institute on Alcohol Abuse and Alcoholism Animal Care and Use Committee.

Isolation and culture of HSCs. Mouse liver HSCs were isolated by in situ collagenase perfusion and differential centrifugation on OptiPrep (Sigma) density gradients, as described previously with some modifications (25). Mouse livers were perfused in situ first with EGTA solution (5.4 mM KCl, 0.44 mM KH2PO4, 140 mM NaCl, 0.34 mM Na2HPO4, 0.5 mM EGTA, 25 mM tricine, pH 7.2), followed by perfusion with perfusion buffer (0.075% collagenase type I in GBSS buffer with 0.02% DNase I) and digestion with digestion buffer (0.009% collagenase type I in Grey's balanced buffer solution buffer with 0.02% DNase I) at 37°C for 20–30 min. The homogenate was filtered and centrifuged at 25 g for 5 min at room temperature to remove the hepatocytes. The supernatant was transferred to a new tube and centrifuged at 400 g for 10 min at 4°C. The cell pellet was then resuspended in 5 ml of 15% OptiPrep, loaded carefully with 5 ml of 11.5% OptiPrep, and centrifuged at 1,400 g for 17 min at 4°C. The cell fraction in the GBSS and 11.5% OptiPrep interphase was gently aspirated, mixed with GBSS, and centrifuged at 1,400 g for 10 min at 4°C. After another wash, the final cell pellet was resuspended in RPMI 1640 medium containing penicillin, streptomycin, and 20% FBS and then plated onto 24-well plates at a density of 1x104 cells per well in 0.5 ml of culture medium or 6-well plates at a density of 1x105 cells per well in 1.5 ml of culture medium. Cell number and viability were assessed by the Trypan blue exclusion test. Viability was always greater than 95%. In each experiment, cells were pooled from four to five mice. Cells were plated onto uncoated plastic plates in all experiments except GoFig. 2C. In Fig. 2C, cells were also plated onto BioCoat Matrigel Matrix Cellware (BD Biosciences, San Jose, CA). After 24 h, nonadherent cells and debris were removed by washing. Purity of the cultures was assessed by typical light microscopic appearance at this point. Vitamin A autofluorescence was more than 90%.


Figure 1
View larger version (33K):
[in this window]
[in a new window]

  		Fig. 1. Hepatic stellate cells (HSCs) derived from C57Bl/6N mice express 5 isoforms of retinoic acid (RA) early inducible gene 1 (RAE-1). A and B: fragments of unique sequences of PCR products amplified with RAE1{alpha} (A) and Raet1{varepsilon} (B) primers are identical to RAE1{alpha} and RAE1{varepsilon} cDNAs, respectively. C–E: agarose gel analyses of the different RAE-1 isoforms. PCR products were amplified with unique RAE-1{varepsilon} forward/pan-RAE-1{varepsilon} reverse primers (C), pan-RAE-1 set1 (D), and pan-RAE-1 set2 (E) primers as described in Table 1, and digested with NcoI or BamHI (C), ApaI plus BamHI (D), and FokI (E), respectively.

 

Figure 2
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 2. Upregulation of RAE-1 mRNA expression in early activated HSCs. A: RT-PCR analyses of the indicated genes in HSCs at different time points postculturing. B: levels of glial fibrillary acidic protein (GFAP) and {alpha}-smooth muscle actin ({alpha}-SMA) mRNA in A were normalized to beta-actin mRNA levels (means ± SD). d, Days. C: HSCs growing on plastic (Pl) or Matrigel (Mg) dishes for the indicated times were subjected to RT-PCR analyses. Pl + Mg means that cells were grown on plastic for 7 days then transferred and grown on Matrigel for an additional 7 days. D: statistical summary of RAE-1 expression in quiescent, early activated, and fully activated HSCs (means ± SD, ***P < 0.001 vs. 0 days).

 
Treatment of cells. For Raldh inhibition experiments, cells were incubated first with 10 µM disulfiram (Sigma) for 2 days and then with 2 µM for an additional 2 days. For retinoid acid receptor (RAR) inhibition, cells were incubated with the RAR antagonist Ro 41-5253 (1) (Biomol International, Plymouth Meeting, PA) at 25 µM for 2 days, then 20 µM for an additional 2 days. The retinoid X receptor (RXR) agonist methoprene acid (15) was purchased from Biomol International. The RAR/RXR agonist all-trans-retinoic acid (ATRA) and the RAR agonist CD437 (4) were purchased from Sigma. Cell culture dishes were light protected during the experiments.

Oil Red O staining. Cytoplasmic fat droplets were stained with 0.5% (wt/vol) Oil Red O (Sigma, St. Louis, MO) in propylene glycol.

RT-PCR. Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA) and cDNA was synthesized with random hexamers by use of SuperScript II Reverse Transcriptase (Invitrogen). Primers used are described in Table 1. For quantitative analysis, the signals generated by the samples were quantified with ImageQuant software (GE Healthcare Life Sciences, Piscataway, NJ) and normalized to beta-actin mRNA levels.


View this table:
[in this window]
[in a new window]

  		Table 1. Primers for PCR

 
Western blotting and FACS analyses of RAE-1 protein. Western blotting was performed as described (18, 30). RAE-1 protein expression on NK cells was also determined by flow cytometric (FACS) analyses by use of RAE-1{gamma} antibody (Bioscience).

RP-HPLC. Cells were quantified and extracted as described (36). Reverse-phase chromatography (RP-HPLC) was carried out on an ÄKTA Purifier HPLC system with a variable-wavelength UV detector (GE Healthcare Life Sciences). The chromatographic conditions were as follows: column, LC-ABZ (15 cm x 4.6 mm ID; particle size, 5 µm; reversed phase C18; Sigma); mobile phase, 57.5% acetonitrile-25% acetic acid (diluted to 2% with water)-17.5% methanol; flow rate, 0.8 ml/min; UV detection wavelength, 354 nm for ATRA and 326 nm for retinol. Acitretin (Sigma) was used as an internal standard. Retinoids were identified by comparing retention volumes. Retinoid concentrations were calculated and referenced against standard curves generated from increasing concentrations of synthetic ATRA or retinol and retinyl palmitate (Sigma). All manipulations with light-sensitive retinoids were carried out under red light.

Cytotoxicity assay. Calcein-AM was purchased from Molecular Probes (Eugene, OR) as a 1 mg/ml solution in dry dimethyl sulfoxide. Cultured HSCs were used as target cells and labeled with 15 µM calcein-AM for 30 min at 37°C with occasional shaking. Liver mononuclear cells were isolated and used as effector cells as described previously (30). The target cells and effector cells (target-to-effector ratio 1:50) were mixed in triplicate in 96-well plates. After 4 h at 37°C in 5% CO2, the supernatant (75 µl) was harvested and calcein-AM fluorescence was measured with a microplate spectrofluorometer (excitation filter: 484 ± 9 nm; brand-pass filter: 530 ± 9 nm). Specific lysis was calculated according to the formula [(test release – spontaneous release)/(maximum release – spontaneous release)] x100.

Statistical analysis. Data are expressed as means ± SD. To compare values obtained from three or more groups, one-factor ANOVA was used, followed by Tukey's post hoc test. To compare values obtained from two groups, the Student's t-test was performed. Statistical significance was taken at the P < 0.05 level.


   RESULTS
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Activated HSCs from C57BL/6n mice express 5 isoforms of RAE-1. It is generally believed that BALB/c mice express three isoforms of RAE-1 ({alpha}, beta, and {gamma}) whereas C57Bl/6 mice express only the {delta} and {varepsilon} isoforms (6). Here we demonstrate, for the first time, that activated HSCs from C57Bl/6N mice express not only RAE-1{delta} and RAE-1{varepsilon}, but also RAE-1{alpha}, RAE-1beta, and RAE-1{gamma}. All five RAE-1 proteins share an 88 to 95% similarity in amino acid identity with only minor differences within the coding regions at the nucleic acid level (6), providing sufficient sequence divergence to discriminate between these isoforms. Thus we performed detailed RT-PCR analyses as well as sequencing and/or restriction enzyme digestion analyses to detect expression of the five different RAE-1 isoforms of activated HSCs obtained from the fibrotic livers of C57Bl/6N mice. The RAE-1{alpha} and RAE-1{varepsilon} sequence alignments indicate that RAE-1{alpha} and RAE-1{varepsilon} can be amplified separately by using forward primers containing unique sequences of corresponding genes (Table 1). Sequencing analyses showed that PCR products amplified with RAE-1{alpha} and RAE-1{varepsilon} primers were identical to RAE-1{alpha} cDNA (Raet1a, GI:667916, positions 221–277) and RAE-1{varepsilon} (Raet1e, GI:38016147, positions 4–15), respectively (Figs. 1, A and B). Further confirmation was obtained for RAE-1{varepsilon} by digestion of the PCR product with NcoI and BamHI restriction enzymes (Fig. 1C). To identify the RAE-1beta, {gamma}, and {delta} isoforms, cDNA prepared from activated HSCs of C57Bl/6N mice were first amplified by pan-RAE-1 primer sets, and then the PCR products obtained were subjected to restriction enzyme digestion. This analysis was based on the presence or absence of specific restriction sites within different RAE-1 isoforms (Table 2). The presence of the 198- and 162-bp bands from PCR products amplified with pan-RAE-1 set1 primers followed by digestion with ApaI and BamHI provided evidence of RAE-1beta and RAE-1{delta} expression, respectively (Fig. 1D). Detection of a 33-bp fragment after digestion of PCR products amplified with pan-RAE-1 set2 primers with the Foci restriction enzyme, which only exists in RAE-1{gamma}, confirmed its expression (Fig. 1E).


View this table:
[in this window]
[in a new window]

  		Table 2. Sequence analysis of RAE-1 cDNA isoforms expression

 
Expression of RAE-1 in early activated HSCs correlates with sensitivity to NK cell killing. To examine the mechanisms by which RAE-1 is upregulated in activated HSCs, we used an in vitro model of cultured HSCs. As shown in Fig. 2, A and B, during early activation (4- and 7-day cultured cells), HSCs coexpressed glial fibrillary acidic protein, a known marker of quiescent HSCs, and {alpha}-smooth muscle actin ({alpha}-SMA), a marker of myofibroblast differentiation. In contrast, fully activated HSCs expressed only {alpha}-SMA. Additionally, early activated HSCs, but not fully activated HSCs (21-day cultured cells), expressed upregulated levels of PDGFRbeta mRNA expression, which is characteristic of early activation and proliferation of HSCs (37). Desmin, recognized as a principal intermediate filament, was detected in both quiescent and activated HSCs. Interestingly, RAE-1 mRNA was detected at high levels in early activated HSCs, but not in quiescent (0-day) or fully activated HSCs. Next, we examined whether inhibition of HSC activation caused by plating cells on Matrigel-containing dishes can prevent RAE-1 induction. As shown in Fig. 2C, cells plated on Matrigel for 7 days (Mg 7d) expressed neither {alpha}-SMA nor RAE-1{alpha}. We also cultured HSCs on petri dishes for 7 days to activate HSCs, and then plated them on Matrigel for another 7 days to deactivate the HSCs. Our results show that RAE-1 mRNA was significantly upregulated in early activated (Pl 7d), but not in deactivated HSCs (Pl + Mg 7d + 7d) (Fig. 2, C and D). These findings suggest that short-term RAE-1 induction is associated with early activation of HSCs.

Expression of RAE-1 proteins was also examined by Western blotting and FACS analyses. As shown in Fig. 3, A and B, RAE-1 protein expression was upregulated in 4- or 7-day cultured HSCs compared with quiescent HSCs. Fig. 3C shows that NK cells produced about 50% cytotoxicity against 4- and 7-day cultured HSCs, but <5% cytotoxicity against quiescent or 21-day cultured HSCs, correlating with RAE-1 expression on these cells. Furthermore, RAE-1 neutralizing antibodies diminished the cytotoxicity of NK cells against HSCs (Fig. 3C).


Figure 3
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 3. Levels of RAE-1 protein expression correlate with the susceptibility of HSCs to natural killer (NK) cell killing. A: Western blot analyses of RAE-1 protein expression in freshly isolated (0 days) and 4-day cultured HSCs. B: FACS analyses of cultured HSCs with RAE-1 antibody. C: cytolytic activity of liver NK cells isolated from polyinosinic-polycytidylic acid treated mice against cultured HSCs preincubated with control IgG or RAE-1 antibody for 30 min. The target-to-effector ratio was 1:25.

 
Induction of RAE-1 correlates with elevation of RA in HSCs. One of the key features of HSC activation is loss of intracellular retinol, which can be metabolized into RA (RA) (3, 14, 28, 31). Interestingly, RAE-1 cDNAs were originally isolated as RA-inducible clones from mouse F9 cells (24). This led us to hypothesize that activation of HSCs may result in a rapid increase in the intracellular concentration of RA, which could in turn upregulate RAE-1. Intracellular retinol is mostly stored as retinyl esters dissolved in fat droplets that are easily visualized by Oil Red O staining. Through Oil Red O staining of fat droplets found in the cytoplasm of HSCs, we showed a correlation between depletion of vitamin A-rich fat droplets (Fig. 4A) and the upregulation of RAE-1 mRNA expression in early activated HSCs (Fig. 2A). Next, we measured the levels of RA and total retinol (retinol plus retinyl ester palmitate) from whole-cell extracts and the supernatant of cultured HSCs. Total intracellular retinol content gradually decreased after culturing and decreased by 80% of its maxima 7 days postculture. In contrast, significant production of ATRA was detected in 4- and 7-day cultured HSCs, whereas no ATRA was detected in quiescent or myofibroblast-like 21-day-old HSCs (Fig. 4B). Significant production of ATRA was also detected in the supernatant of 4-day, but not 1-day, cultured HSCs (Fig. 4C). These results indicate that early HSC activation is accompanied by endogenous synthesis of RA, coinciding with the upregulation of RAE-1 expression.


Figure 4
View larger version (51K):
[in this window]
[in a new window]

  		Fig. 4. Production of RA in early activated HSCs. A: Oil Red O staining of early activated HSCs (4- and 7-day). Arrows indicate red-stained fat droplets. B: concentrations of intracellular retinol and RA in cultured HSCs were measured by reverse-phase (RP)-HPLC. C: Concentrations of RA in the supernatant from 1- and 4-day cultured HSCs were measured. ATRA, all-trans-retinoic acid.

 
Inhibition of Raldhs abolishes induction of RAE1 in early activated HSCs. Figure 5A shows the expression of Raldh1 and Raldh2 mRNA, which are essential for RA synthesis in HSCs (14). The Raldh1 mRNA was slightly induced, whereas Raldh2 mRNA was markedly induced in 4- and 7-day cultured HSCs (Fig. 5A), correlating with RA accumulation in these cells (Fig. 4B). Blocking Raldhs with disulfiram, a potent inhibitor of RA synthesizing enzymes (34), abolished RA production (Fig. 5B). Interestingly, treatment with disulfiram also diminished expression of RAE-1{alpha}/{varepsilon} and pan-RAE-1 as well as Raldh1 and Raldh2 mRNA (Fig. 5C). Inhibition of Raldh1/2 mRNA by disulfiram is likely due to the fact that disulfiram treatment blocks RA production that is responsible for induction of Raldh mRNA expression in a positive feedback loop (38). In addition to inhibition of Raldhs, disulfiram has also been shown to attenuate proteasome activity and nuclear NF-{kappa}B translocation (19). To rule out the possibility that disulfiram inhibits RAE1 expression via mechanisms other than inhibition of Raldhs, we examined whether ATRA treatment was able to reverse disulfiram-mediated inhibition of RAE1 mRNA. Fig. 5D shows that disulfiram inhibition of RAE1 mRNA expression was partially reversed by treatment with ATRA, suggesting that disulfiram inhibits RAE-1 mRNA expression via at least in part inhibition of RA production.


Figure 5
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 5. Inhibition of retinol dehydrogenases (Raldhs) abolishes induction of RAE1 in early activated HSCs. A: expression of Raldh1/2 mRNA was examined by RT-PCR in cultured HSCs. B: RP-HPLC analyses of RA in conditioned medium from control and disulfiram-treated HSCs. I.S., internal standard acitretin. C: Raldh1/2 and RAE-1 isoform mRNA expression in disulfiram-treated and untreated HSCs were examined by RT-PCR. D: freshly isolated HSCs were cultured for 2 days with disulfiram (10 µM) and then changed to medium containing disulfiram (2 µM) with or without ATRA for an additional 2 days. RAE1 mRNA was determined by RT-PCR.

 
Involvement of RA signaling in RAE-1 upregulation in early activated HSCs. RA activity is mediated via binding to the nuclear retinoid receptors, RAR ({alpha}, beta, {gamma}) and RXR ({alpha}, beta, {gamma}) (22). Figure 6, A and B, shows that RAR {alpha}, beta, and {gamma} and RXR {alpha} and beta transcripts were detectable at high levels in HSCs during the first week of culture followed by a gradual decrease in expression in 21-day cultured cells. In contrast, retinoic acid binding protein (Crabp), a negative regulator of the biological activity of RA, was not induced in HSCs after culturing (data not shown). Treatment with the RAR antagonist, Ro 41-5253 suppressed RAE-1 induction in HSCs during activation (Fig. 6C), whereas treatment with RAR/RXR agonist ATRA or RAR agonist CD437 (4) for 2 days resulted in significant induction of RAE-1{alpha} and pan-RAE1 mRNA expression (Fig. 6D). Treatment with RXR agonist methoprene acid (15) did not induce RAE-1{alpha} mRNA expression but induced pan-RAE1 mRNA expression.


Figure 6
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 6. Requirement of RA signaling for RAE-1 upregulation in activated HSCs. A: expression of retinoic acid receptor (RAR)/retinoic X receptor (RXR) isoforms was examined by RT-PCR in cultured HSCs. B: quantitative analyses of RT-PCR data from A. C: RAE-1 mRNA expression in RAR antagonist Ro-41-4253-treated and untreated HSCs. D: freshly isolated HSCs were cultured for 2 days with RAR/RXR agonist ATRA (5 µM), RAR agonist CD437 (10 µM), or RXR agonist methoprene acid (MA) (10 µM). Expression of RAE1 mRNA was determined by RT-PCR.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
So far, five RAE1 isoforms ({alpha}, beta, {gamma}, {delta}, and {varepsilon}) have been identified. Among them, expression of {delta} and {varepsilon} isoforms but not {alpha}, beta, and {gamma} isoforms was reported in C57Bl/6 mice whereas BALB/c mice express only {alpha}, beta, and {gamma} isoforms (6). By using RT-PCR, sequencing, and restriction enzyme digestion analyses, here we demonstrated, for the first time, that activated HSCs from C57Bl/6N mice express all five isoforms of RAE-1 (Fig. 1). These findings suggest that C57Bl/6N mice do express RAE-1{alpha}, beta, and {gamma} isoforms under diseased conditions such as liver fibrosis. Moreover, we investigated the mechanisms underlying RAE1 upregulation in activated HSCs by using an in vitro culture model. Our findings showed that, similar to in vivo, early activated HSCs from in vitro culture also had elevated expression of RAE1, and RA produced by HSCs during activation is responsible for RAE1 induction. We have integrated these findings in a model in Fig. 7 and the rationale for this model is discussed below.


Figure 7
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 7. Model for how RA signaling initiates NK cell killing of early activated HSCs. Quiescent HSCs store 75% of the body's supply of retinol (vitamin A). During the early stages of activation, retinol can be metabolized into retinal through dehydrogenase 1 (ADH1) and ADH3 and subsequently metabolized into RA through Raldh1 and Raldh2. RA induces expression of RAE-1. As an activating ligand for NK cells, RAE-1 initiates NK cell killing of early activated HSCs through releasing TNF-related apoptosis-inducing ligand (TRAIL), which targets TRAIL receptors (TRAILR) that are upregulated in activated HSCs (33). After fully activated, HSCs lose retinol and do not express RAE-1, thereby becoming resistant to NK killing.

 
HSCs play a key role in intrahepatic uptake, storage, and release of retinoids (5, 14). During the activation, HSCs lose intracellular retinol and produce to RA via ADHs and Raldhs (3, 5, 14, 31, 32). Here we provide several lines of evidence suggesting that production of RA by HSCs during early activation likely contributes to induction of RAE1 expression in these cells. First, production of RA by HSCs during early activation at days 4 and 7 (Fig. 4) correlated with induction of RAE1 expression in these cells (Fig. 2). Second, inhibition of RA synthesizing enzyme Raldhs with disulfiram blocked RA production and diminished RAE1 expression (Fig. 5). Although disulfiram also inhibits proteasome activity and nuclear NF-{kappa}B translocation in addition to blocking Raldhs and RA synthesis (19), RA treatment was able to reverse partially disulfiram-mediated inhibition of RAE1 mRNA (Fig. 5D), suggesting that disulfiram inhibition of RA synthesis contributes to disulfiram inhibition of RAE1 expression. However, we cannot rule out other mechanisms may also contribute to disulfiram inhibition of RAE1 expression because only partial reversal by ATRA was observed. Finally, expression of RA receptors (RARs and RXRs) was induced in early activated HSCs. Treatment of HSCs with RAR antagonist Ro 41-5253 inhibited RAE1{alpha}, RAE1{varepsilon}, and pan-RAE-1 expression whereas treatment with RAR/RXR agonist ATRA or RAR agonist CD437 (4) stimulated RAE1{alpha} and pan-RAE-1 expression, suggesting that activation of RAR is involved in induction of RAE1{alpha}, {varepsilon}, and other forms of RAE1. Treatment with RXR agonist methoprene acid (15) only induced pan-RAE-1 but not RAE-1{alpha} mRNA expression, indicating that activation of RXR is not involved in induction of RAE-1{alpha} but contributes to induction of other forms of RAE-1. Although our findings clearly show that RA is responsible for induction of RAE-1 in cultured HSCs during activation, whether it also contributes to RAE-1 induction in activated HSCs in vivo during liver fibrogenesis is not clear. It has been reported that, similar to in vitro culture, RA levels were significantly elevated in the liver during fibrogenesis induced by CCl4, thioacetamide, or porcine serum (23, 27) models of liver injury, but not by bile duct ligation (26). The reason for the discrepancy between these models is not clear. Since elevation of hepatic RA was observed only in the early stages of liver fibrosis (1 and 2 mo post-CCl4 treatment), but not the late stages (after 4 mo of CCl4 treatment) (23), and production of RA was also diminished in fully activated HSCs, it is plausible that HSCs in the bile duct ligation model at 19 days postsurgery (26) have already become completely activated and lost all of its retinol, resulting in low levels of RA. Indeed, diminished RA signaling was found in HSCs obtained after 19-day but not 7- to 10-day bile duct ligation (H. Tsukamoto personal communication), suggesting that HSCs from 19-day bile duct ligation are fully activated since RA signaling is decreased only in fully activated HSCs (Fig. 4). Collectively, RA is likely produced and induces RAE-1 expression in HSCs in the early stages, but not late stages of liver fibrosis in vivo.

RAE1 was previously identified as an NK cell activating ligand to stimulate NK cytotoxicity (6). Here we also confirmed that RAE1 expression on activated HSCs plays an important role in initiating NK cell killing of these cells. As shown in Fig. 3, early activated HSCs that express high levels of RAE1 were susceptible to NK cell killing whereas quiescent or fully activated HSCs that lack expression of RAE1 were resistant to such killing. RAE1 neutralizing antibody diminished the cytotoxicity of NK cells against HSCs (Fig. 3), further supporting the critical role of RAE1 in NK cell killing of HSCs. Another important finding in the paper is that fully activated HSCs lost RAE1 expression and became resistant to NK killing (Figs. 2 and 3). This is because fully activated HSCs not only lose production of RA but also become irresponsive to RA stimulation owing to downregulation of RAR and RXR expression (Fig. 6) (16). Although our in vitro evidence shows that RAE-1 induction contributes to the susceptibility of activated HSCs to NK cell killing, the biological significance of RAE-1 induction in activated HSCs in vivo is unclear. It is plausible that such induction also contributes to NK cell killing of activated HSCs because, after depletion of NK cells, the number of activated HSCs is increased in the fibrotic livers due to less HSC apoptosis (Fig. 3A in Ref. 30 and our unpublished data).

In summary, RA signaling sensitizes HSCs to NK cell killing via induction of RAE-1 during early activation. This process may contribute to the antifibrotic effect of RA in the liver (35), in addition to the direct inhibition of collagen synthesis and HSC proliferation by RA (16, 26). After being fully activated, HSCs become nonresponsive to RA signaling and resistant to NK cell killing, which could lead to chronic liver fibrosis and disease.


   GRANTS
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the intramural program of the National Institute of Alcohol Abuse and Alcoholism.


   ACKNOWLEDGMENTS
 
We thank the National Institutes of Health (NIH) Fellow Editorial Board for editorial assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Radaeva or B. Gao, Section on Liver Biology, NIAAA/NIH, 5625 Fishers Lane, Rm. 2S-33, Bethesda, MD 20892 (e-mail: sradaeva{at}mail.nih.gov or bgao{at}mail.nih.gov)

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
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Apfel C, Bauer F, Crettaz M, Forni L, Kamber M, Kaufmann F, LeMotte P, Pirson W, Klaus M. A retinoic acid receptor alpha antagonist selectively counteracts retinoic acid effects. Proc Natl Acad Sci USA 89: 7129–7133, 1992.[Abstract/Free Full Text]
  2. Backstrom E, Chambers BJ, Ho EL, Naidenko OV, Mariotti R, Fremont DH, Yokoyama WM, Kristensson K, Ljunggren HG. Natural killer cell-mediated lysis of dorsal root ganglia neurons via RAE1/NKG2D interactions. Eur J Immunol 33: 92–100, 2003.[CrossRef][Web of Science][Medline]
  3. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 115: 209–218, 2005.[CrossRef][Web of Science][Medline]
  4. Bernard BA, Bernardon JM, Delescluse C, Martin B, Lenoir MC, Maignan J, Charpentier B, Pilgrim WR, Reichert U, Shroot B. Identification of synthetic retinoids with selectivity for human nuclear retinoic acid receptor gamma. Biochem Biophys Res Commun 186: 977–983, 1992.[CrossRef][Web of Science][Medline]
  5. Blomhoff R, Wake K. Perisinusoidal stellate cells of the liver: important roles in retinol metabolism and fibrosis. FASEB J 5: 271–277, 1991.[Abstract]
  6. Cerwenka A, Lanier LL. Ligands for natural killer cell receptors: redundancy or specificity. Immunol Rev 181: 158–169, 2001.[CrossRef][Web of Science][Medline]
  7. Chen Y, Wei H, Sun R, Dong Z, Zhang J, Tian Z. Increased susceptibility to liver injury in hepatitis B virus transgenic mice involves NKG2D-ligand interaction and natural killer cells. Hepatology 2007 Jul 11; [Epub ahead of print].
  8. Duester G. Families of retinoid dehydrogenases regulating vitamin A function: production of visual pigment and retinoic acid. Eur J Biochem 267: 4315–4324, 2000.[Web of Science][Medline]
  9. Duester G. Function of alcohol dehydrogenase and aldehyde dehydrogenase gene families in retinoid signaling. Adv Exp Med Biol 463: 311–319, 1999.[Web of Science][Medline]
  10. Elsharkawy AM, Oakley F, Mann DA. The role and regulation of hepatic stellate cell apoptosis in reversal of liver fibrosis. Apoptosis 10: 927–939, 2005.[CrossRef][Web of Science][Medline]
  11. Fowell AJ, Iredale JP. Emerging therapies for liver fibrosis. Dig Dis 24: 174–183, 2006.[CrossRef][Web of Science][Medline]
  12. Friedman SL, Bansal MB. Reversal of hepatic fibrosis—Fact or fantasy? Hepatology 43: S82–S88, 2006.[CrossRef][Web of Science][Medline]
  13. Friedman SL, Rockey DC, Bissell DM. Hepatic fibrosis 2006: report of the Third AASLD Single Topic Conference. Hepatology 45: 242–249, 2007.[CrossRef][Web of Science][Medline]
  14. Geerts A. History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells. Semin Liver Dis 21: 311–335, 2001.[CrossRef][Web of Science][Medline]
  15. Harmon MA, Boehm MF, Heyman RA, Mangelsdorf DJ. Activation of mammalian retinoid X receptors by the insect growth regulator methoprene. Proc Natl Acad Sci USA 92: 6157–6160, 1995.[Abstract/Free Full Text]
  16. Hellemans K, Verbuyst P, Quartier E, Schuit F, Rombouts K, Chandraratna RA, Schuppan D, Geerts A. Differential modulation of rat hepatic stellate phenotype by natural and synthetic retinoids. Hepatology 39: 97–108, 2004.[CrossRef][Web of Science][Medline]
  17. Iredale JP. Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. J Clin Invest 117: 539–548, 2007.[CrossRef][Web of Science][Medline]
  18. Jeong WI, Park O, Radaeva S, Gao B. STAT1 inhibits liver fibrosis in mice by inhibiting stellate cell proliferation and stimulating NK cell cytotoxicity. Hepatology 44: 1441–1451, 2006.[CrossRef][Web of Science][Medline]
  19. Lovborg H, Oberg F, Rickardson L, Gullbo J, Nygren P, Larsson R. Inhibition of proteasome activity, nuclear factor-KappaB translocation and cell survival by the antialcoholism drug disulfiram. Int J Cancer 118: 1577–1580, 2006.[CrossRef][Web of Science][Medline]
  20. Mehal WZ. Activation-induced cell death of hepatic stellate cells by the innate immune system. Gastroenterology 130: 600–603, 2006.[CrossRef][Web of Science][Medline]
  21. Melhem A, Muhanna N, Bishara A, Alvarez CE, Ilan Y, Bishara T, Horani A, Nassar M, Friedman SL, Safadi R. Anti-fibrotic activity of NK cells in experimental liver injury through killing of activated HSC. J Hepatol 45: 60–71, 2006.[CrossRef][Web of Science][Medline]
  22. Napoli JL. Retinoic acid biosynthesis and metabolism. FASEB J 10: 993–1001, 1996.[Abstract]
  23. Natarajan SK, Thomas S, Ramachandran A, Pulimood AB, Balasubramanian KA. Retinoid metabolism during development of liver cirrhosis. Arch Biochem Biophys 443: 93–100, 2005.[CrossRef][Web of Science][Medline]
  24. Nomura M, Takihara Y, Shimada K. Isolation and characterization of retinoic acid-inducible cDNA clones in F9 cells: one of the early inducible clones encodes a novel protein sharing several highly homologous regions with a Drosophila polyhomeotic protein. Differentiation 57: 39–50, 1994.[CrossRef][Web of Science][Medline]
  25. Oben JA, Yang S, Lin H, Ono M, Diehl AM. Acetylcholine promotes the proliferation and collagen gene expression of myofibroblastic hepatic stellate cells. Biochem Biophys Res Commun 300: 172–177, 2003.[CrossRef][Web of Science][Medline]
  26. Ohata M, Lin M, Satre M, Tsukamoto H. Diminished retinoic acid signaling in hepatic stellate cells in cholestatic liver fibrosis. Am J Physiol Gastrointest Liver Physiol 272: G589–G596, 1997.[Abstract/Free Full Text]
  27. Okuno M, Sato T, Kitamoto T, Imai S, Kawada N, Suzuki Y, Yoshimura H, Moriwaki H, Onuki K, Masushige S, Muto Y, Friedman SL, Kato S, Kojima S. Increased 9,13-di-cis-retinoic acid in rat hepatic fibrosis: implication for a potential link between retinoid loss and TGF-beta mediated fibrogenesis in vivo. J Hepatol 30: 1073–1080, 1999.[CrossRef][Web of Science][Medline]
  28. Pinzani M, Rombouts K. Liver fibrosis: from the bench to clinical targets. Dig Liver Dis 36: 231–242, 2004.[CrossRef][Web of Science][Medline]
  29. Purohit V, Brenner DA. Mechanisms of alcohol-induced hepatic fibrosis: a summary of the Ron Thurman Symposium. Hepatology 43: 872–878, 2006.[CrossRef][Web of Science][Medline]
  30. Radaeva S, Sun R, Jaruga B, Nguyen VT, Tian Z, Gao B. Natural killer cells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent manners. Gastroenterology 130: 435–452, 2006.[CrossRef][Web of Science][Medline]
  31. Reeves HL, Friedman SL. Activation of hepatic stellate cells—a key issue in liver fibrosis. Front Biosci 7: d808–d826, 2002.[Web of Science][Medline]
  32. Senoo H. Structure and function of hepatic stellate cells. Med Electron Microsc 37: 3–15, 2004.[CrossRef][Medline]
  33. Taimr P, Higuchi H, Kocova E, Rippe RA, Friedman S, Gores GJ. Activated stellate cells express the TRAIL receptor-2/death receptor-5 and undergo TRAIL-mediated apoptosis. Hepatology 37: 87–95, 2003.[CrossRef][Web of Science][Medline]
  34. Vallari RC, Pietruszko R. Human aldehyde dehydrogenase: mechanism of inhibition of disulfiram. Science 216: 637–639, 1982.[Abstract/Free Full Text]
  35. Wang L, Potter JJ, Rennie-Tankersley L, Novitskiy G, Sipes J, Mezey E. Effects of retinoic acid on the development of liver fibrosis produced by carbon tetrachloride in mice. Biochim Biophys Acta 1772: 66–71, 2007.[Medline]
  36. Williams JB, Napoli JL. Metabolism of retinoic acid and retinol during differentiation of F9 embryonal carcinoma cells. Proc Natl Acad Sci USA 82: 4658–4662, 1985.[Abstract/Free Full Text]
  37. Wong L, Yamasaki G, Johnson RJ, Friedman SL. Induction of beta-platelet-derived growth factor receptor in rat hepatic lipocytes during cellular activation in vivo and in culture. J Clin Invest 94: 1563–1569, 1994.[Web of Science][Medline]
  38. Zhao D, McCaffery P, Ivins KJ, Neve RL, Hogan P, Chin WW, Drager UC. Molecular identification of a major retinoic-acid-synthesizing enzyme, a retinaldehyde-specific dehydrogenase. Eur J Biochem 240: 15–22, 1996.[Web of Science][Medline]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
B. Gao, S. Radaeva, and O. Park
Liver natural killer and natural killer T cells: immunobiology and emerging roles in liver diseases
J. Leukoc. Biol., September 1, 2009; 86(3): 513 - 528.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/4/G809    most recent
00212.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Radaeva, S.
Right arrow Articles by Gao, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Radaeva, S.
Right arrow Articles by Gao, B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.