DHRS3, a retinal reductase, is differentially regulated by retinoic acid and lipopolysaccharide-induced inflammation in THP-1 cells and rat liver

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1 Am J Physiol Gstrointest Liver Physiol 33: G78 G88, 212. First pulished July 12, 212; doi:1.112/jpgi DHRS3, retinl redutse, is differentilly regulted y retinoi id nd lipopolyshride-indued inflmmtion in THP-1 ells nd rt liver Rez Zolfghri, Qiuyn Chen, nd A. Cthrine Ross Deprtment of Nutritionl Sienes nd Huk Institute for Life Sienes, Pennsylvni Stte University, University Prk, Pennsylvni Sumitted 8 June 212; epted in finl form 1 July 212 Zolfghri R, Chen Q, Ross AC. DHRS3, retinl redutse, is differentilly regulted y retinoi id nd lipopolyshride-indued inflmmtion in THP-1 ells nd rt liver. Am J Physiol Gstrointest Liver Physiol 33: G78 G88, 212. First pulished July 12, 212; doi:1.112/jpgi Both retinoid sttus nd inflmmtion hve een shown to ontrol the level of expression of retinoid homeostti genes. In the present study, DHRS3, previously shown to possess retinl redutse tivity, ws identified y mirorry nlysis of THP-1 monoytes s possile gene trget of ll-trns-retinoi id (RA). In these ells, DHRS3 mrna inresed 3- to -fold fter tretment with 2 nm RA for 2 h, while DHRS3 protein lso inresed. Of severl syntheti retinoids tested, only Am8, RA reeptor- -seletive retinoid, inresed DHRS3 mrna expression. The full-length DHRS3 DNA ws loned from rt liver nd sujeted to in vitro trnsription-trnsltion. Two mjor 3- nd 3-kD proteins were deteted. In dult rt tissues, DHRS3 mrna ws most undnt in the drenl glnd, liver, nd ovry. In the liver, DHRS3 is expressed in heptoytes nd possily in ll liver ells. To evlute whether DHRS3 is regulted in the liver y RA nd/or inflmmtory stimuli, we treted rts for 6 h with RA or LPS or oth. DHRS3 mrna ws douled y RA ut redued y 9% fter tretment with LPS in the sene nd presene of RA. On the sis of our results, DHRS3 mrna expression is regulted y RA in tissue- or ell-type speifi mnner; the RA-indued inrese in DHRS3 my ontriute to retinoid storge; nd redution of DHRS3 expression in the liver during inflmmtion my ontriute to the perturtion of whole ody vitmin A metolism tht hs previously een shown to our in onditions of inflmmtory stress. inflmmtion; -rotene metolism; liver; retinoid metolism Address for reprint requests nd other orrespondene: A. C. Ross, The Pennsylvni Stte Univ., 11 Chndlee Lortory, Univ. Prk, PA 1682 (e-mil: r6@psu.edu). THE BIOCHEMICAL REACTIONS nd regultory proesses tht mintin vitmin A (retinoid) homeostsis re inompletely understood. Retinl, the ldehyde form of vitmin A, is intrinsilly retive euse of the ility of its ronyl group to form Shiff ses with proteins, phospholipids, or retinl itself (38). Additionlly, its iologil oxidtion produt, ll-trns-retinoi id (RA), is potent hormone ple of regulting gene trnsription through inding to nuler retinoid reeptors (1, 13, ). Retinl is metolized intrellulrly through redutive nd oxidtive retions. All-trns-retinl is not only n intermedite in the oxidtion proess tht onverts retinol to RA, ut it is lso the first intermedite in the oxidtive levge of -rotene (2, 2, 2, 2), nutritionl preursor of vitmin A. One retinl is redued enzymtilly to retinol, the retinol my e esterified with long-hin ftty ids nd stored. In the retin, the light-tlyzed lehing of 11-is-retinl lso produes ll-trns-retinl, whih is enzymtilly redued to lltrns-retinol in the visul proess (3). Although high onentrtions (3 mm) of retinl hve een reported in the outer segment of photoreeptor ells (1), the onentrtion of retinl in the liver nd most peripherl tissues is mintined t muh lower levels. The onversion of retinl to retinol hs een reported to e medited through severl retinol dehydrogenses (29, 3, 36), of whih the well-onserved mirosoml protein DHRS3, memer of the lssil short-hin dehydrogense redutse (SDR) superfmily, hs een previously identified (1). Although DHRS3 ws originlly loned from the retin, it is expressed in severl non-oulr humn tissues, inluding the liver, pnres, hert, kidney, nd lung (3, 1). In ell extrts prepred from HEK-293T ells trnsfeted with DHRS3 DNA, retinl ws redued to retinol in n NADPH-dependent mnner (1). In neurolstom ell lines trnsfeted with DHRS3, the metolism of retinl or retinol to retinyl ester ws fvored (3). During emryoni development, whih is ritilly dependent on finely regulted onentrtions of RA (3), DHRS3 ws required in the emryo, primrily within the entrl nervous system (12), where it pprently funtions s n RA-indued feedk inhiitor of RA synthesis nd is highly tive t the onset of gstrultion (17). The DHRS3 gene mps to region of humn hromosome 1 t nd p36.1 (1), whih is frequently lost in ggressive neurolstom tumors (3). DHRS3 expression ws reported to e indued y retinoids in neurolstom ell lines (3) nd in nïve CD T ells (19) nd y the retinoid X reeptor (RXR) rexinoid lignd exrotene in MMTV-erB2 mie (23) nd to e responsive to serum onentrtion in norml nd trnsformed humn ul kertinoytes (39). Reent studies hve shown tht DHRS3 is regulted y p3 nd p63 tumor suppressor proteins (2) nd is le to promote lipid droplet storge in HepG2 ells, onsistent with the loliztion of DHRS3 in the endoplsmi retiulum (8). Little is known out the physiologil regultion of DHRS3 expression in vivo. Severl enzymes in the liver tht hve een implited in the regultion of retinol metolism hve een shown to e regulted y RA, inluding leithin:retinol yltrnsferse (LRAT), nd CYP26 fmily genes (33). Conversely, the expression of these genes ws rpidly downregulted fter the indution of inflmmtion in rt model of LPS-indued ute inflmmtion (), while inflmmtion lso interfered with retinol trnsport in plsm (1). However, it is not known whether inflmmtion lso lters the oxidtive metolism of retinol y DHRS3. Using two models, the humn monoyti ell line THP-1, whih hs een shown to undergo differentition in response to RA s well s ute response to inflmmtory stimuli (, 6), nd rt liver, whih is relevnt to the regultion of whole ody retinoid homeostsis G /12 Copyright 212 the Amerin Physiologil Soiety

2 nd to the ute-phse response to infetion (31), we hve tested the hypothesis tht DHRS3 expression is regulted y RA nd inflmmtion. MATERIALS AND METHODS Regents. All-trns-RA, 9-is-RA, olei id, tinomyin D, nd yloheximide were purhsed from Sigm-Aldrih (St. Louis, MO). TNF ws otined from R&DSystems (Minnepolis, MN). LPS from Pseudomons eruginos ws purhsed from Biologil Lortory (Cmpell, CA) nd dissolved in PBS efore use. Reeptorseletive retinoids were kindly provided y Mihel Klus (Hoffmnn-L Rohe, Nutley, NJ). Stok solutions of retinoids were mde in solute ethnol, nd olei id ws dissolved diretly in serumfree ell ulture medium. RPMI 16 medium, DMEM, het-intivted FBS, nd TRIzol regent were purhsed from Invitrogen Life Tehnology (Crlsd, CA). Cell ulture. THP-1 ells were grown s previously desried in RPMI 16 medium () supplemented with 1% het-intivted FBS, whih ws redued to 3% FBS for 2 h prior to tretment. Tretments inluded inution with 2 nm RA in ethnol (finl onentrtion.1%), ng/ml of TNF (, 28), or 1 ng/ml of LPS (, 28), lone or in omintion, for 6 h. In seprte experiments, the ells were lso treted with reeptor-seletive retinoids (2 nm eh), inluding Am8 [ RA reeptor (RAR)- (RAR ) gonist], Ro19-6 ( RAR gonist), CD37 ( RAR gonist), nd Ro ( RXR pn-gonist). DHRS3 gene expression in THP-1 ells ws deteted y mirorry nlysis, s desried previously for gltose mutrotse (ldose 1-epimerse) (GALM) gene expression (28), using totl RNA from ells treted with 2 nm RA, without or with ng/ml of TNF, under the onditions desried ove. Humn heptom HepG2 ells were grown in six-well pltes in the presene of DMEM with 1% FBS. Upon rehing 8% onfluene, the ells were treted with 1 M RA for,., 1, 2,, nd 6hnd then wshed nd olleted for RNA isoltion with TRIzol regent. Animl tretments. Animl protools were pproved y the Animl Cre nd Use Committee of Pennsylvni Stte University. Briefly, vitmin A-dequte dult Sprgue-Dwley rts (Chrles River Lortories, Wilmington, MA) were fed vitmin A-dequte diet () nd treted with single dose of g of RA, delivered orlly in 3 l of vegetle oil, whih ws lso used s the pleo ontrol, or with single injetion of P. eruginos LPS ( g/1 g ody wt ip), or oth, s previously reported (). After 6 h, the rts were killed y CO 2 inhltion, lood smples were drwn for preprtion of plsm, nd tissues were olleted nd immeditely snp-frozen for RNA isoltion (). Liver smples from rts tht hd een fed the sme diet lking vitmin A to produe stte of vitmin A defiieny were lso used; the rts were then treted with single dose of 1 g ofrain time-ourse study, s desried previously (32, 33). Rts were euthnized, nd the liver ws olleted t time (vehile ontrol) nd 3, 6, 1, nd 16 h fter RA dministrtion. RNA isoltion nd nlysis. Totl RNA ws isolted from individul tissue smples with TRIzol Regent. Poly(A) RNA ws isolted from totl RNA nd sujeted to grose gel eletrophoresis nd trnsferred to Nytrn memrnes, whih were hyridized with fulllength rt 32 P-leled DNA proes (). The memrnes were wshed nd then exposed to Kodk BioMx X-ry film (Estmn Kodk, Rohester, NY). Cloning of rt liver DHRS3 DNA. A pir of primers ws designed from the predited sequene ville in the GenBnk dtse ( nd used to lone the = nd 3= ends of the DHRS3 mrna y rpid mplifition of DNA ends (RACE), following the strtegy reported previously (7), using poly(a) - enrihed RNA of rt liver from RA-treted rts. The =- nd 3=-RACE produts were loned y TA loning (pgem-t Esy vetor, Promeg, Mdison, WI) nd sujeted to sequening. Upon lignment, the G79 sequene of the full-length DHRS3 DNA lone ws ssemled nd deposited to the GenBnk dtse (ession no. EF12189). In vitro trnsription nd trnsltion of rt DHRS3. On the sis of the sequene of the full-length rt DHRS3 DNA (ession no. EF12189), pir of primers, inluding =-ttggtggcaatca- GATCGCGTTTAA-3= (XhoI site in lowerse letters) s the sense nd =-tttgattcctgccgcgtgaccagt-3= (XI site in lowerse letters) s the ntisense, were designed nd used to mplify DNA overing lmost the entire region (2,2 p) of DHRS3 DNA y RT-PCR from rt liver totl RNA, s desried previously (7). The mplified DNA ws first loned in pgem-t Esy vetor nd then suloned into the XhoI/XI site of the pdna3.1( ) vetor (Invitrogen Life Tehnology). The pdna3.1( ) vetor ontining the rt DHRS3 lone ws then used s templte in n in vitro trnsription-trnsltion ssy using the Single Tue Protein System 3 kit (Novgen, EMD Chemils, Gistown, NJ) in the presene of [ 3 S]methionine (GE Helthre Bio-Siene, Pistwy, NJ) following the mnufturer s instrutions extly. A 1- l liquot of the retion produt ws sujeted to eletrophoresis in n SDS-polyrylmide gel (12%) under reduing onditions to size-frtionte the leled protein produts. The gel ws dried under vuum nd then exposed to X-ry film (Estmn Kodk, Rohester, NY). Plsmid DNA for preprtion of RNA proes. The rt DNA frgment overing nt 17 1 of the rt DHRS3 DNA ws suloned from pgem-t Esy vetor ontining DHRS3 DNA into the SmI site of the pgem vetor. The pgem-rdhrs3 lone ws then linerized with EoRI or XI nd used s templte to synthesize ntisense or sense RNA proes with T7 or SP6 RNA polymerse, respetively. The rt retinol-inding protein (RBP) DNA (8 p) in the pgem vetor (32) ws linerized with NrI or HindIII nd used s the templte to synthesize the ntisense or sense RNA proe y T7 or SP6 RNA polymerse, respetively. On the sis of the sequene of rt hemokine (C-X-C motif) lignd 1 (melnom growth-stimulting tivity-, CXCL1) mrna (ession no. NM_38), pir of primers, inluding =-AAACCAGCTCCAGCACTCC-3= s the sense nd =TTTCATTTGTAACAGTCCTTTGAA-3= s the ntisense, were designed nd used to mplify DNA overing lmost the entire region of rt CXCL1 DNA (9 p) y RT-PCR from totl RNA of the rts treted with LPS s desried. The mplified DNA ws first loned in the pgem-t Esy vetor nd then suloned into the SmI site of the pgem vetor. The pgem-rcxcl1 lone ws then linerized with EoRI or XI nd used s templte to synthesize ntisense or sense RNA proes, respetively, s desried ove. For eh proe, digoxigenin (DIG)-leled sense or ntisense RNA rioproe ws prepred using the DIG RNA leling kit in retion with rionuleotides inluding DIG-UTP (Rohe Biotehnology). The leled RNA ws isolted, heked for size y ethidium romide-stined grose gel eletrophoresis, nd then treted in lkline solution to prepre 1- to 1-se polynuleotide lengths s proe (32). In situ hyridiztion. Frozen liver setions (1 m thik) were pled on glss slides, fixed with % prformldehyde, nd hyridized in % formmide uffer solution with DIG-leled rioproe t 2 C overnight, s previously desried (32). After they were wshed nd loked, the slides were inuted overnight with lkline phosphtse (AP)-onjugted nti-dig ntiody or horserdish peroxidse-onjugted nti-dig ntiody (Rohe Biotehnology, Indinpolis, IN) in loking uffer overnight (32). After the slides treted with AP-onjugted ntiody were wshed, they were inuted with -romo--hloro-3-indolyl phosphte-nitro lue tetrzolium s syntheti sustrte for AP (Rohe Biotehnology) nd then evluted under digitl olor mirosope in our lortory. The slides treted with horserdish peroxidse-onjugted ntiody were mplified (Tyrmine Signl Amplifition Plus Fluoresein System, Perkin Elmer nd Life Anlytil Sienes) following the protool provided y the mnufturer, nd the resulting fluorophores were deteted y fluo-

3 G8 resene visuliztion using the fluoresene mirosope in our lortory. Quntifition of DHRS3 mrna. The mrna level of DHRS3 nd other trnsripts in ells nd in individul tissue smples ws determined y quntittive RT-PCR () using totl RNA prepred s desried ove. The PCR yling progrm ws first set t 9 C for 1 min to tivte the Tq polymerse nd then t yles of 9 C for 1 s, 6 C for 3 s, nd 72 C for 3 s, with sense nd ntisense primers s follows: =-ACTTTCCACCTCCGGCTTCC-3= nd =-CGTTCTCCCGCGA- CAGGTC-3= for humn DHRS3 (GenBnk ession no. NM_73), =-TTGTCCACCGCCTCCTACCT-3= nd =-GTGCCCATCTGC- CGAATCT-3= for rt DHRS3 (GenBnk ession no. EF12189), =-CACGGGGCTTTGTGTCTATT-3= nd =-CCTCGTCGTAAT- GAGGGTGT-3= for rt -rotene 1,1=-monooxygense 1 (GenBnk ession no. NM_368), =-CGGACCCATTTTACCCACTA-3= nd =-CAGACTGCAGGAAGGGTCAT-3= for rt LRAT (GenBnk ession no. AF26), nd =-CGCGGTTCTATTTTGTTGGT-3= nd =-AGTCGGCATCGTTTATGGTC-3= for 18S rrna (GenBnk ession no. X1117). Eh RNA trnsript ws mesured seprtely nd lulted using 18S rrna s n internl ontrol. Dt were normlized to the verge vlue for the ontrol group, set t 1., efore sttistil nlysis. Immunolot nlysis. Cells wshed in PBS nd lysed in RIPA uffer were prepred, nd equl mounts (3 6 g) of lyste protein were sujeted to SDS-PAGE, trnsferred to nitroellulose memrne, nd inuted with nti-humn DHRS3 monolonl ntiody (kindly provided y Dr. Krzysztof Plzewski, Cse Western Reserve University Shool of Mediine, Clevelnd, OH) nd then with seondry ntiody, s desried previously (6). For loding ontrols, the memrnes were stined for protein with Poneu solution. Sttistil nlysis. Vlues re mens SE. Dt were nlyzed y one-wy ANOVA with Fisher s post ho test. RESULTS DHRS3 mrna nd protein re rpidly inresed y RA in THP-1 ells. When THP-1 ells were treted with onentrtion of RA typil of levels found in plsm (2 nm) (26), DHRS3 mrna expression, determined y mirorry nlysis, ws signifintly upregulted fter 2 h, inresed further y 6 h, nd then remined stle for up to h (Fig. 1A), inditing 16-fold inrese in the level of DHRS3 mrna. In seond study designed to determine the response of THP-1 ells to RA nd TNF, oth of whih hve een shown to indue THP-1 ell differentition towrd mrophge-like phenotype (), RA (2 nm) ws signifint ftor for DHRS3 mrna expression (Fig. 1B), wheres TNF ( ng/ml) lone hd no effet. The presene of TNF did not lter the response of THP-1 ells to RA (Fig. 1B). To verify the mirorry results of DHRS3 mrna expression, we performed two seprte experiments on THP-1 ells: we mesured DHRS3 mrna y quntittive PCR nd protein y immunolot nlysis. In the first experiment, DHRS3 mrna, whih ws rely deteted in the ontrol ells (Fig. 1C), ws highly expressed fter 6 h (Fig. 1C, inset) to2h (Fig. 1C) of tretment with 2 nm RA. Similrly, DHRS3 protein ws not detetle in the ontrol ells; however, protein nd tht orresponded to the size of DHRS3 ws signifintly expressed in RA-treted THP-1 ells within 6 h (Fig. 1D). In seond experiment, THP-1 ells were treted with 2 nm RA in the presene or sene of TNF ( ng/ml) or LPS (1 ng/ml) for 2 h. Wheres DHRS3 mrna ws inresed 3-fold y RA (Fig. 1C nd inset), neither TNF nor LPS hd signifint effet on the level of DHRS3 mrna in these ells, in the sene or presene of RA. Similr results were otined for DHRS3 protein levels nlyzed y immunolot nlysis (Fig. 1E). Agonist-seletive indution of DHRS3 mrna. RA is onsidered pn-gonist lignd for ll RAR sutypes, inluding RAR, RAR, nd RAR. To determine whih reeptor sutype(s) is involved, diretly or indiretly, in the indution of DHRS3 mrna expression y RA, we treted THP-1 ells with vrious gonists tht hve een shown to e seletive for inding to nd tivting memers of the RAR fmily. Of severl retinoids tested, only Am8, retinoid with gretest ffinity for RAR (9, ), ws s effetive s RA in induing DHRS3 mrna expression in THP-1 ells (Fig. 2A). Retinoids seletive for inding to RAR (Ro19-6) nd RAR (CD37) hd no signifint effet, lthough these reeptors re expressed in this ell line (28); 9-is-RA, lignd ple of tivting RAR nd RXR sutypes (18), produed smll ut signifint effet, while pn-rxr lignd (Ro2-7386) hd no signifint effet on expression of DHRS3 mrna (Fig. 2A). Olei id, used s lipophili ompound with physiohemil properties similr to RA, lso ws ineffetive (Fig. 2A). The ility of RA to inrese the expression of DHRS3 mrna is likely to e t the trnsriptionl level, s the inrese in DHRS3 mrna fter RA tretment ws ompletely prevented in ells pretreted with tinomyin D ( g/ml) for 6 h (Fig. 2B). On the other hnd, yloheximide, n inhiitor of protein synthesis, loked the inresed expression fter RA tretment signifintly, ut not ompletely (Fig. 2C). However, yloheximide hs previously een shown to ttenute the indution y RA of prtiulr form of DHRS3 trnsript in neurolstom ells (3). DHRS3 is expressed in vrious tissues of the rt. Next, we exmined whether suh regultion ours in the whole orgnism. We seleted the rt euse of the numerous similrities in the sorption, trnsport, nd metolism of retinol etween rts nd humns. After loning, lignment, nd ssemly of the rt DHRS3 DNA (see MATERIALS AND METHODS), DNA lone with 2,111 ses ws otined (GenBnk ession no. EF12189). Unlike the humn DHRS3 mrna previously reported (1), the rt DHRS3 mrna ontins n ATG loted 68 ses upstrem of the predited ATG, ut not in the sme reding frme. Therefore, to verify the size of the predited rt DHRS3 protein, we suloned the nerly full-length DNA sequene into the pdna3.1 vetor nd used n in vitro trnsription-trnsltion ssy with [ 3 S]methionine inorportion for detetion of the newly trnslted protein. After the 3 S-leled protein produt ws sujeted to SDS-PAGE, two mjor 3- to 3-kD nds were oserved on the film (Fig. 3A), inditing tht the downstrem ATG t position 76 my e onsidered the initition odon for n open reding frme similr to tht of humn DHRS3 (1) nd ruling out the upstrem ATG s n effetive initition odon in this ssy. On this sis, the rt DHRS3 mrna ontins n open reding frme of 99 ses with =- nd 3=-untrnslted regions of 7 nd 7 ses, respetively. Rt DHRS3 mrna is signifintly longer thn humn DHRS3 mrna, yet the numer of mino id residues is similr to tht of humn nd mouse DHRS3 protein, with 9% nd 99% mino id identity, respetively ( gov/homologene; Fig. 3B).

4 G81 A B DHRS3 signl intensity (log 2) >>,P <. RA, h: D C DHRS3 mrna, reltive to ontrol RA Con RA E TNFα DHRS3 signl intensity (log 2) RA (2 nm) LPS RA RA/ TNFα RA TNFα RA,TNFα RA/ LPS DHRS3 18S TNFα LPS RA/TNFα RA/LPS Fig. 1. Expression nd regultion of DHRS3 mrna in THP-1 ells treted with retinoi id (RA), TNF, or LPS, nd omintions of these gents. A: DHRS3 mrna in THP-1 ells treted with 2 nm RA t time ; n equl mount of RA ws dded fter 2 h of inution. B: DHRS3 mrna fter h of tretment with RA, TNF ( ng/ml, dded 16 h fter time ), nd RA (dded t time ) followed y TNF (dded t 16 h for totl of h). Results in A nd B represent 2 6 replites, determined y mirorry nlysis. For 2 different trget sequenes of DHRS3 mrna, log 2 differene (mximum minimum) ws.16 (A) nd.16 (not shown), respetively. C: reltive intensity of DHRS3 mrna in THP-1 ells treted with vehile or RA (2 nm), eh in triplite, for 6 h nd then hrvested for RNA nlysis y semiquntittive RT- PCR. Inset: ethidium romide-stined grose gel. Tretments were s follows: vehile only (Con), RA (2 nm), TNF ( ng/ml), LPS (1 ng/ml), RA TNF, nd RA LPS (n 3/group). D: DHRS3 protein deteted y immunolot on pooled ell extrts using humn DHRS3 monolonl ntiody. THP-1 ells were ultured with vehile only (ontrol) nd with RA (2 nm, 6 h). Poneu S stining ws used s loding ontrol. E: DHRS3 protein deteted y immunolot using humn DHRS3 monolonl ntiody. Cell extrts were prepred from THP-1 ells treted s desried in C. Poneu S stining ws used s loding ontrol. Differenes were determined y 1-wy ANOVA: different letters (,, ) indite signifint differene: P.. DHRS3 Poneu S stining To investigte the tissue distriution of DHRS3 mrna, totl RNA smples from eh tissue of four different dult rts were individully sujeted to reverse trnsription followed y quntittive PCR nlysis (see MATERIALS AND METHODS). DHRS3 is expressed in most tissues (Fig. 3C), with the highest reltive undne of DHRS3 mrna trnsripts in the drenl glnd, liver, nd ovry; intermediry level of expression in the smll intestine, kidney, nd lung; nd lower level of expression in the dipose tissue, hert, mmmry tissue, testis, rin, nd eye. Rt DHRS3 mrna is pprently sent from or expressed t only very low levels in the pnres. In n in situ hyridiztion ssy, rt liver setions in glss slides were hyridized to DIG-leled ntisense or sense rioproes for DHRS3, RBP, nd CXCL1 nd then nlyzed y fluoresene (for DHRS3) or olor detetion system (for RBP nd CXCL1). Wheres signifint signls were deteted in the liver setions y the ntisense rioproes (Fig. 3D, imges,, nd e), no detetle signls were oserved with the sense rioproes (Fig. 3D, imges, d, nd f). DHRS3 mrna is pprently expressed in heptoytes (Fig. 3D, imge ), lthough not s exlusively s RBP mrna, the heptoytespeifi gene (Fig. 3D, imge ), whih hs een reported y us (32) nd others (37) to hve strong expression nd heptoyte-exlusive distriution. As nonprenhyml ell-speifi gene, we used CXCL1, whih hs een reported to e expressed in neutrophils nd Kupffer ells in response to liver injury (16). We found no signl for CXCL1 in the setion of the liver of norml rts (dt not shown), ut in the liver of rts treted with LPS, CXCL1 ws deteted in isolted ells or smll lusters ppering s mononuler ells (Fig. 3D, imge e).

5 G82 A Fig. 2. DHRS3 mrna expression is regulted y RA reeptor (RAR)-speifi retinoids, tinomyin D, nd yloheximide in THP-1 ells. A: ells were treted in triplite with vrious RAR sutype-speifi nd retinoid X reeptor (RXR)-speifi retinoids (2 nm) for 6 h. Reeptor lignds were s follows: Am8 for RAR, Ro19-6 for RAR, CD37 for RAR, nd Ro s pn-gonist for RXR. tra, ll-trns- RA. B: ells were treted with tinomyin D (AtD, g/ml) for 1 h efore ddition of RA (2 nm) for nother 6 h. C: ells were treted with yloheximide (CHX, g/ml) for 1 h efore ddition of RA (2 nm) for nother 6 h. After eh experiment, RNA ws nlyzed y quntittive RT-PCR for DHRS3 mrna nd 18S RNA, nd results were normlized. Different letters indite signifint differene (P.). B DHRS3 mrna, reltive to ontrol DHRS3 mrna, reltive to ontrol At. D tra 9isRA RARα RARβ RARγ RXR Olei Lignds id RA At. D +RA C DHRS3 mrna, reltive to ontrol CHX RA CHX +RA DHRS3 expression hs een reported in humn heptom HepG2 ells (8), well-differentited ell model for heptoytes (21). In ell ulture experiments, we mesured DHRS3 mrna levels in HepG2 ells treted without or with RA for up to 6 h. DHRS3 mrna is omprtively expressed in HepG2 ells (verge yle numer for detetion ws in HepG2 ells vs in THP-1 ells), nd its expression is indued fter h of tretment with RA y out two- to threefold (dt not shown), whih is muh lower thn tht in THP-1 ells. DHRS3 expression in rt liver is orrelted with tht of LRAT. Hving oserved tht DHRS3 mrna is highly expressed in the liver, the mjor site of vitmin A storge (3), we wished to determine if DHRS3 is regulted y vitmin A sttus or RA tretment in this orgn. Thus we nlyzed rt liver RNA smples () from dult vitmin A-dequte rts nd similr rts treted with RA or low dose of LPS to indue inflmmtion (31) or oth. Blood nd tissues were olleted fter 6 h. These short-term tretments did not lter the onentrtion of retinol in plsm or liver (). Totl RNA extrted from individul liver smples ws sujeted to quntittive RT-PCR nlysis for expression of DHRS3, s well s -rotene monooxygense (BCMO1) nd LRAT, the genes tht puttively my t upstrem nd downstrem of DHRS3, respetively (Fig. A). In rts treted with RA lone, DHRS3 mrna inresed to out twie the ontrol level (Fig. B; P.). Although this inrese ws signifint, it ws muh less thn the 3-fold inrese in RA-treted THP-1 ells. However, in ontrst to the results for THP-1 ells in whih LPS tretment hd no effet on DHRS3 expression, in vivo tretment with LPS redued the expression of DHRS3 mrna in the liver y 9% (P.1) in the sene nd presene of RA (Fig. B). Although the indution effet of RA ws low in the expression of DHRS3 in the liver, it ours pprently through trnsriptionl proess similr to tht oserved in THP-1 ells (Fig. 2B), sine tretment of the individul rts with tinomyin D, lone or with RA, for 6 h suppressed the expression of DHRS3 mrna in the liver y 9% (P.1; Fig. C). This suggests tht the stedy-stte level nd RA-indued inrese in DHRS3 mrna re minly due to the trnsriptionl proess. We lso mesured BCMO1 nd LRAT, s genes involved 1) in prodution of retinl from -rotene nd 2) in removl of the puttive produt of the DHRS3 retion, retinol. After vitmin A-dequte rts were treted with RA or LPS for 6 h, the level of BCMO1 mrna in the liver ws redued y % (P.; Fig. D). However, dministrtion of RA nd LPS together did not use further redution. We lso nlyzed the expression of LRAT, whih tlyzes the esterifition of retinol, the puttive produt of the enzymti retion of DHRS3. Tretment of vitmin A-dequte rts with RA inresed LRAT mrna in the liver y 2.-fold (P.; Fig., E nd F). In ontrst to tretment with RA, tretment with LPS, lone or in omintion with RA, ttenuted the expression of LRAT mrna signifintly (P.) y 8% (Fig., E nd F). As expression levels of DHRS3 nd LRAT mrna re upregulted y RA nd downregulted y LPS t lmost the sme mgnitude in the liver of norml rts, we found strong orreltion etween DHRS3 mrna levels nd those of LRAT ross severl experiments (R 2.83, P.1, n 2). Reently, we reported the kineti response to in vivo tretment with RA for severl retinoid homeostti genes, inluding LRAT, known to e responsive to RA (7), in the liver of rts first mde defiient in vitmin A nd then given single dose of RA (32). Gene expression ws mesured t seline (defiient stte) nd 3, 6, 1, nd 16 h fter dministrtion of RA. LRAT mrna inresed signifintly in the first 3 h, rehed pek t 6 1 h, nd then delined. In the urrent study, we used the sme liver smples to exmine the response of

6 G83 Fig. 3. In vitro trnsription of loned rt DHRS3 nd expression of DHRS3 mrna in vrious tissues of dult rts. A: in vitro trnsription nd trnsltion of rt liver DHRS3. Full-length DHRS3 DNA ws isolted from rt liver nd loned into pdna3.1 vetor for in vitro trnsription-trnsltion using rit retiuloyte lyste in the presene of [3S]methionine. PAGE ws onduted under denturing onditions for seprtion of the 3S-leled protein produts. Gel ws dried under vuum t 8 C nd exposed to X-ry film, s desried elsewhere (7). ORF, open reding frme. B: lignment of protein sequene of rt DHRS3 (rdhrs3) with humn nd mouse DHRS3 (hdhrs3 nd mdhrs3). Protein sequene of rt DHRS3 (ession no. NP_132276) ws ligned with humn DHRS3 (ession no. NP_7) nd mouse DHRS3 (ession no. NP_333) sequenes using Rt DHRS3 protein is 9% identil to humn nd 99% identil to mouse DHRS3 protein ( C: reltive expression level of DHRS3 mrna in different tissues of rts. Totl RNA from individul tissues of dult rts ws sujeted to quntittive RT-PCR for DHRS3 mrna nlysis. Vlues re mens SE of different rts reltive to vlue for liver, whih is onsidered 1.. Vlues for drenl glnd nd ovry re from pooled RNA smples. D: loliztion of DHRS3 mrna in liver of rts y in situ hyridiztion. Liver setions (1 m) in glss slides were hyridized to digoxigenin-leled ntisense (imges,, nd e) or sense (imges, d, nd f) rioproes for DHRS3 (imges nd ), retinol-inding protein (RBP; imges nd d), nd CXCL1 (imges e nd f) nd then nlyzed y fluoresene for DHRS3 or olor detetion system for RBP nd CXCL1. Arrows in imge e indite site of expression of CXCL1, whih ppered s isolted mononuler ells in liver setions of rts treted with LPS. DHRS3 mrna levels nd the orreltion of its expression to the response of LRAT (32). DHRS3 mrna (Fig. A) followed nerly the sme kinetis s those of LRAT, with more thn fourfold inrese within the first 3 h, plteu to 6 h, nd then deline. DHRS3 mrna ws signifintly nd strongly orrelted with LRAT (R2.9, P.6). To evlute gene expression profile in the liver under different dietry regimens, we estlished onditions tht produe

7 G8 Fig.. Regultion of DHRS3, -rotene 1,1=-monooxygense 1 (BCMO1), nd leithin:retinol yltrnsferse (LRAT) mrna expression in liver of dult rts treted in vivo with RA, LPS, or oth. A: shem of pthwy involving genes metolizing retinl. RE, retinyl ester; REH, RE hydrolse; RDH, retinol dehydrogense; RALDH, retinldehyde dehydrogense. B, D, nd F: totl RNA ws extrted from individul liver smples (n 6/group) nd then mesured y rel-time quntittive RT-PCR for DHRS3, BCMO1, or LRAT, s well s for 18S RNA s the ontrol. B: DHRS3 expression in liver of dult rts fed vitmin A-dequte diet nd treted with RA ( g/kg ody wt), LPS ( g/kg ody wt) (), or oth for 6 h. C: effet of tinomyin D (At D) on expression of DHRS3 mrna in liver of dult rts. Rts were treted with tinomyin D lone or with RA for 6 h, nd liver tissues were olleted for totl RNA extrtion nd nlysis. Vlues re mens SE (n 3/group). D: BCMO1 mrna from smples used in B. E: Northern lot nlysis of LRAT mrna from liver of rts treted s desried in B. ATRA, ll-trns-ra. F: quntittive RT-PCR for LRAT mrna from smples used in B. Different letters indite signifint differene: P. for B, D, nd F; P.1 for C. RE A B LRAT REH DHRS3 mrna, reltive to ontrol C DHRS3 mrna reltive to ontrol β-crotene BCM1 Retinol DHRS3 RALDH Retinl Retinoi id RDH RA LPS RA/LPS At D RA At D + RA D BCMO1 mrna, reltive toontrol E F LRAT mrna, reltive to ontrol RA LPS RA/LPS 28S 18S 28S 18S ATRA LPS ATRA/LPS ATRA LPS ATRA/LPS RA LPS RA/LPS wide rnge of vitmin A sttus from defiient to mrginl, dequte, nd supplemented vitmin A sttus in growing helthy rts (32). As n inditor of vitmin A sttus, the verge liver totl retinol in vitmin A-defiient, -mrginl, nd -supplemented groups ws.3,.3, nd times tht in the vitmin A-dequte group, respetively (32). Although DHRS3 mrna expression orrelted well with the totl retinol levels in the liver, the mrna levels were not signifintly lower in defiient nd mrginl nimls or not signifintly higher in supplemented rts thn in vitmin A-dequte ontrol rts. However, the verge expression levels of DHRS3 mrna were lower in vitmin A-defiient thn vitmin A-supplemented rts (P.; Fig. B). DHRS3 mrna expression levels were orrelted with those of LRAT (R 2.72, P.1), s previously reported (32). Comprison with ldo-keto redutse expression. Severl ldo-keto redutse (AKR) enzymes, whih re ytoplsmi proteins, from sufmily 1B nd 1C hve een reported to hve tlyti tivity towrd the redution of retinl to retinol (36). Among them, humn AKR1B1 nd hiken AKR1B12 hve een shown to hve muh higher tlyti tivities towrd redution of ll-trns-retinl, wheres humn AKR1C3 hve een reported to hve muh higher preferene for the redution of 9-is-retinl, s the retinoid sustrte. However, rt AKR1B1, whether or not it is onsidered the humn ortholog, hs een shown to hve slight tlyti tivity towrd redution of ll-trns-retinl (11). It is not known whether AKR1B7 hs tlyti tivity towrd redution of retinls. In our gene expression profile ssy, we found tht, mong the AKR genes, only the expression level of AKR1B7 mrna is inresed y 7% (P.) y RA in the liver of vitmin A-defiient rts (Fig. 6A), ut not in vitmin A-suffiient nimls (Fig. 6B). As omprison to DHRS3, the AKR1B7 gene ws suppressed slightly, ut signifintly (P.), in the liver of

8 G8 A Log 2 of Signl Intensity B DHRS3 mrna (log 2) nimls treted with LPS (Fig. 6B). We found no signifint hnges in the expression of AKR1B1 or AKR1B1 gene in the liver of rts treted with RA or LPS (dt not shown). DISCUSSION DHRS3 RA, h: , In the present study, we first found tht DHRS3 expression is highly regulted y RA in THP-1 mononuler ells, model used in numerous studies of monoyti ell differentition (). Among the RAR- nd RXR-seletive retinoids used, only Am8, retinoenzoi id nlog of RA tht seletively inds to nd tivtes RAR (9) nd lso hs een shown to indue ell differentition (9, ), ws highly effetive in inresing the expression of DHRS3 mrna. Whether this ours diretly nd is medited y lignd-tivted RAR or indiretly through other genes hs not een estlished; however, the indution of DHRS3 y RA seems to our t the trnsriptionl level, euse 1) the indution ws reltively fst, exemplified in our studies y the nerly eightfold inrese in DHRS3 mrna within 2 h fter tretment of THP-1 ells with physiologil onentrtion of RA (26) nd within 3 h, VAD VAM VA-d VASup Fig.. Reltive mrna levels of DHRS3 in liver of dult rts. A: kinetis of regultion of DHRS3 expression in liver of vitmin A-defiient rts fter single olus dose of RA. Livers of vitmin A-defiient rts were nlyzed, 3, 6, 1, nd 16 h following injetion of single olus dose of 1 g RA, s previously reported (32). B: reltive mrna levels of DHRS3 mrna in liver of rts fed vitmin A-defiient (VAD), -mrginl (VAM), -dequte (VA-d), nd -supplemented (VASup) diets from wening to 8 wk of ge. Vlues re mens SE; n, 7, 6, nd 2 rts for VAD, VAM, VA-d, VASup, respetively. Different letters indite signifint differene: P.. fter tretment of vitmin A-defiient rts with RA in vivo nd 2) the indution of DHRS3 mrna expression y RA in THP-1 ells, s well s in the liver of rts, ws inhiited ompletely y tretment with tinomyin D, known inhiitor of trnsription. Similr results hve een reported in neurolstom ell lines when doxoruiin, nother inhiitor of the trnsriptionl proess, ws used (3). On the sis of physil nlysis of the DNA sequene of DHRS3 gene, we found diret repet-2- like RA response element loted 1. kp upstrem of the ATG odon. This diret repet-2 element is pprently onserved in the humn, mouse, nd rt. Whether suh n element hs ny responsive funtion to vitmin A remins to e determined. To ompre the results oserved in THP-1 ells with physiologilly relevnt in vivo model, we exmined the expression of this gene in the rt. We first loned the full-length DNA from the liver nd ompred it with tht of humn DHRS3, whih ws previously reported (1). Although rt DHRS3 mrna ppers to e longer thn humn DHRS3 mrna, oth ode for proteins of similr size, with highly identil (9%) mino id residues ( nih.gov/homologene) (Fig. 3B). As shown y quntittive RT-PCR, the rt DHRS3 mrna is highly expressed in the drenl glnd, liver, nd ovry ut is less undnt in our rt tissue thn in the previously reported humn tissue lot for few tissues, inluding the pnres, whih showed stronger expression for humn DHRS3 mrna (3, 1). This enzyme is likely to ply n importnt role in vitmin A nd -rotene A AKR17 mrna (log 2) B AKR17 mrna (log 2) Hours fter RA dministrtion RA LPS RA/LPS >>, P<. Fig. 6. Reltive mrna levels of ldo-keto redutse (AKR1B7) in liver of dult rts. A: liver smples from rts studied in Fig. A. B: liver smples from rts studied in Fig. B. Vlues re mens SE for eh group. Different letters indite signifint differene: P..

9 G86 metolism, whih differ somewht etween the humn nd rt in their digestive proessing. Alterntively, retinl redutses other thn DHRS3 my e present in the rt digestive system. In this regrd, sustntil retinl redutse tlyti tivity ws reported in the homogente of intestinl tissues of young rts (27). DHRS3 is pprently expressed in heptoytes, lthough not s exlusively s RBP (Fig. 3D) nd CYP26A1 (32), eh onsidered heptoyte-speifi genes. As demonstrted during emryoni development (12), DHRS3 nd CYP26A1 my t defensively to protet ginst uildup of RA in heptoytes, where oth re expressed. As in THP-1 ells, DHRS3 mrna expression in the liver of RA-treted vitmin A-dequte rts ws upregulted y RA. However, the indution of DHRS3 expression in this se ws 2-fold, ompred with 3- to -fold in THP-1 ells. Prt of this differene my e due to the higher sl expression of DHRS3 mrna in the liver. The physiologil stte of the niml ould lso e signifint ftor, s rts mde modertely defiient in vitmin A responded rpidly nd strongly to RA dministrtion, with signifint inrese in DHRS3 mrna within 3 h, plteu to 6 h, nd deline to the seline y 16 h (Fig. ). RA, whih is known to turn over rpidly in vivo, is not stored; thus it is not surprising tht the indution of DHRS3 mrna ws trnsient. However, the dt lso suggest tht the hlf-life of DHRS3 mrna is reltively short. The more drmti regultion of DHRS3 expression y RA in the vitmin A-defiient liver is onsistent with tht of other retinoid-metolizing genes, suh s ytohrome P- genes, espeilly CYP26A1 nd CYP26B1, whih re downregulted in the vitmin A-defiient stte ut expressed t reltively higher levels in the vitmin A-dequte stte nd respond rpidly to RA (32, 33). Thus RA my e lered very quikly upon entry into the liver of the vitmin A-dequte host, therey limiting the response of potentilly RA-regulted genes. Indeed, metolism of RA in the liver of rts tht were treted with olus intrvenous dose of [ 3 H]RA in vivo ws signifintly slower in vitmin A-defiient thn vitmin A-dequte rts (7). The mrna for LRAT, the enzyme ultimtely responsile for vitmin A storge (32), inresed in similr mnner to DHRS3 mrna in vitmin A-defiient, s well s vitmin A-suffiient, rts in this study. The orreltion etween DHRS3 mrna (Fig. ) nd LRAT mrna (32) lso ws very strong nd signifint. These dt lso support the possiility of onerted regultion of BCMO1, DHRS3, nd LRAT (Fig. ) tht, together, ould promote retinyl ester formtion nd ensure the sequestrtion of ny exess retinol produed from the levge of -rotene to retinl, followed y the tions of DHRS3 on retinl to form retinol nd LRAT to form retinyl esters. The finding tht DHRS3 mrna is severely downregulted in the liver of LPS-treted rts is lso of physiologil interest. The model of LPS-indued inflmmtion used in our studies is reltively mild, hving een shown to result in smll inrese of 1 C in ore ody temperture within 6 h, redued food intke, nd moderte shivering (31); however, rts reovered well from this tretment within 2 3 dys. The dose we hve used, g/kg ody wt, is muh lower thn tht used in mny studies of LPS-indued liver pthology. In our in vivo studies in norml rts, tretment with LPS nerly ompletely extinguished DHRS3 mrna expression in the liver, with or without otretment with RA. This result ontrsted to the result otined in THP-1 ells in whih LPS hd no effet on DHRS3, despite its well-doumented effets on other proesses (). The results with LPS re lso of interest regrding -rotene metolism. The liver is reognized site of signifint postintestinl -rotene levge (1). If BCMO1 is onsidered sole provider of sustrte, retinl, for the DHRS3 enzyme, then it is possile tht, under onditions of LPSindued inflmmtion, retinl would umulte, nd perhps similrly in sttes of nturl infetion or inflmmtion. It is well known tht free retinl is very unstle ompound tht my result in formtion of free rdils, whih re ple of omining with proteins nd phospholipids (38) to form ompounds tht re elieved to e highly dmging to host tissues, unless the tivity of BCMO1 is lso suppressed or the tivity of retinl dehydrogense(s) is t lest mintined upon LPS tretment. In the present study, the level of BCMO1 mrna in the liver of LPS-treted rts ws lso redued, lthough not to the extent to whih DHRS3 mrna ws redued (Fig. ). It is possile tht differenes in the rte of deline of DHRS3 nd BCMO1 mrnas fter LPS tretment ould e due to intrinsi differenes in the turnover rtes of the preexisting mrnas for eh gene or to other differenes in mrna. The results of our study suggest tht LPS ould indue n inrese in the turnover of DHRS3 nd BCMO1 mrnas in rt liver. The results lso suggest tht there ould e some signifint uildup of retinl during LPS tretment, unless retinl is further oxidized to RA through retinl dehydrogense(s) or tolized in some other mnner. The strong orreltion etween DHRS3 mrna nd LRAT mrna we oserved seems to suggest oordinted proess involved in DHRS3 nd LRAT gene expression in the liver during LPS tretment. DHRS3 hs een shown to e expressed in only those rest ner ell lines tht were ple of esterifying retinol, ut not in other ell lines tht lked retinol esterifition pity (3). Moreover, inution of stly DHRS3-trnsfeted neurolstom ell lines or RA-treted neurolstom ell lines with retinl or retinol resulted in more esterifition of retinol (3). Sine LRAT is reognized s the mjor enzyme responsile for retinol esterifition, this my indite oordintion of the funtions of DHRS3 nd LRAT. Finlly, DHRS3 my not e the only enzyme in the liver to tlyze the redution of retinl to retinol. There my e others, inluding AKR1B or AKR1C, s well s other retinol dehydrogenses/redutses (29, 3, 36). Among AKR1B sufmily enzymes, we found tht only the AKR1B7 gene is indued y RA in the liver of vitmin A-defiient, ut not vitmin A-suffiient, diet-fed rts. Compred with the DHRS3 gene, whih is suppressed lmost entirely in the liver of rts treted with LPS, the AKRB1B7 mrna level is slightly ut signifintly redued. The AKR1B7 enzyme is not known, however, to hve tlyti tivity towrd redution of retinl. In summry, the present experiments hve demonstrted tht the expression of DHRS3 mrna is differentilly regulted in vitro in THP-1 ells nd in vivo in the liver of dult rts. Wheres RA mrkedly indued DHRS3 mrna expression in THP-1 ells, the upregultion of DHRS3 in rt liver in vivo depended quntittively on the retinoid nutritionl sttus of the host. Inflmmtion, whih is reognized s entrl ftor in mny, if not ll, hroni diseses, resulted in the lmost omplete suppression of the expression of DHRS3 mrna in

10 the liver of dult rts, even opposing the indution y RA. The suppression of DHRS3 mrna in the liver in sttes of inflmmtion, suh s were indued y LPS in the present study, my ontriute to the perturtion of vitmin A metolism, inluding loss of stored vitmin A (22) nd redued rte of seretion of retinol from liver into plsm (1), s hs een shown to our during infetion nd inflmmtion. GRANTS This work ws supported in prt y Ntionl Institutes of Helth Grnts CA-921 nd DK-179. DISCLOSURES No onflits of interest, finnil or otherwise, re delred y the uthors. AUTHOR CONTRIBUTIONS R.Z., Q.C., nd A.C.R. re responsile for oneption nd design of the reserh; R.Z. nd Q.C. performed the experiments; R.Z., Q.C., nd A.C.R. nlyzed the dt; R.Z., Q.C., nd A.C.R. interpreted the results of the experiments; R.Z. nd A.C.R. prepred the figures; R.Z. drfted the mnusript; R.Z., Q.C., nd A.C.R. edited nd revised the mnusript; R.Z., Q.C., nd A.C.R. pproved the finl version of the mnusript. REFERENCES 1. Bstien J, Rohette-Egly C. Nuler retinoid reeptors nd the trnsription of retinoid-trget genes. Gene 328: 1 16, Bieslski HK, Chihili GR, Frnk J, von Lintig J, Nohr D. Conversion of -rotene to retinl pigment. Vitm Horm 7: , Cerignoli F, Guo X, Crdinli B, Rinldi C, Csletto J, Frti L, Srepnti I, Guds LJ, Gulino A, Thiele CJ, Ginnini G. retsdr1, short-hin retinol dehydrogense/redutse, is retinoi id-induile nd frequently deleted in humn neurolstom ell lines. Cner Res 62: , 22.. Chen Q, M Y, Ross AC. Opposing ytokine-speifi effets of ll trns-retinoi id on the tivtion nd expression of signl trnsduer nd tivtor of trnsription (STAT)-1 in THP-1 ells. Immunology 17: , 22.. Chen Q, Ross AC. Retinoi id regultes CD1d gene expression t the trnsriptionl level in humn nd rodent monoyti ells. Exp Biol Med (Mywood) 232: 88 9, Chen Q, Ross AC. Retinoi id regultes ell yle progression nd ell differentition in humn monoyti THP-1 ells. Exp Cell Res 297: 68 81, Cifelli CJ, Ross AC. All-trns-retinoi id distriution nd metolism in vitmin A-mrginl rts. Am J Physiol Gstrointest Liver Physiol 291: G19 G22, Deisenroth C, Ithn Y, Tollini L, Jin A, Zhng Y. p3-induile DHRS3 is n endoplsmi retiulum protein ssoited with lipid droplet umultion. J Biol Chem 286: , Delesluse C, Cvey MT, Mrtin B, Bernrd BA, Reihert U, Mignn J, Drmon M, Shroot B. Seletive high ffinity retinoi id reeptor or lignds. Mol Phrmol : 6 62, Dowling JE. The retin: n pprohle prt of the rin. Struture 1: 9 91, Endo S, Mtsung T, Kurgno T, Ohno S, Kitde Y, Tjim K, El-Kni O, Hr A. Properties nd tissue distriution of novel ldo-keto redutse enoding in rt gene (Akr11). Arh Biohem Biophys 3: , Feng L, Hernndez RE, Wxmn JS, Yelon D, Moens CB. Dhrs3 regultes retinoi id iosynthesis through feedk inhiition mehnism. Dev Biol 338: 1 1, Fields AL, Soprno DR, Soprno K Jr. Retinoids in iologil ontrol nd ner. J Cell Biohem. 1. Gieng SH, Green MH, Green JB, Rosles FJ. Model-sed omprtmentl nlysis indites redued moiliztion of hepti vitmin A during inflmmtion in rts. J Lipid Res 8: 9 913, Heseleer F, Hung J, Leiod L, Sri JC, Plzewski K. Moleulr hrteriztion of novel short-hin dehydrogense/redutse tht redues ll-trns-retinl. J Biol Chem 273: , G Hrty MW, Pp EF, Huddleston HM, Young E, Nzreth S, Riley CA, Rmm GA, Gregory SH, Try TFJ. Hepti mrophges promote the neutrophil-dependent resolution of firosis in repiring holestti rt livers. Surgery 13: , Km RK, Chen Y, Chn SO, Chn WY, Dwid IB, Zho H. Developmentl expression of Xenopus short-hin dehydrogense/redutse 3. Int J Dev Biol : , Kne MA. Anlysis, ourrene, nd funtion of 9-is-retinoi id. Biohim Biophys At 1821: 1 2, Kng SG, Prk J, Cho JY, Ulrih B, Kim CH. Complementry roles of retinoi id nd TGF- 1 in oordinted expression of muosl integrins y T ells. Muosl Immunol : 66 82, Kirshner RD, Rother K, Müller GA, Engelnd K. The retinl dehydrogense/redutse retsdr1/dhrs3 gene is tivted y p3 nd p63 ut not y mutnts derived from tumors or EEC/ADULT mlformtion syndromes. Cell Cyle 9: , Knowles BB, Howe CC, Aden DP. Humn heptoellulr rinom ell lines serete the mjor plsm proteins nd heptitis B surfe ntigen. Siene 29: 97 99, Li D, Friedmn SL. Liver firogenesis nd the role of hepti stellte ells: new insights nd prospets for therpy. J Gstroenterol Heptol 1: , Li Y, Zhng Y, Hill J, Kim HT, Shen Q, Bissonnette RP, Lmph WW, Brown PH. The rexinoid, exrotene, prevents the development of premlignnt lesions in MMTV-erB2 mie. Br J Cner 98: , Lietz G, Lnge J, Rimh G. Moleulr nd dietry regultion of, -rotene 1,1=-monooxygense 1 (BCMO1). Arh Biohem Biophys 2: 8 16, Npoli JL. Physiologil insights into ll-trns-retinoi id iosynthesis. Biohim Biophys At 1821: , Npoli JL, Prmnik BC, Willims JB, Dwson MI, Hos PD. Quntifition of retinoi id y gs-liquid hromtogrphy-mss spetrometry: totl versus ll-trns-retinoi id in humn plsm. J Lipid Res 26: , Ong DE, Lus PC, Kkkd B, Quik TC. Ontogeny of two vitmin A-metolizing enzymes nd two retinol-inding proteins present in the smll intestine of the rt. J Lipid Res 32: , Pi T, Chen Q, Zhng Y, Zolfghri R, Ross AC. Gltomutrotse nd other gltose-relted genes re rpidly indued y retinoi id in humn myeloid ells. Biohemistry 6: , Prés X, Frrés J, Kedishvili N, Duester G. Medium- nd short-hin dehydrogense/redutse gene nd protein fmilies: medium-hin nd short-hin dehydrogenses/redutses in retinoid metolism. Cell Mol Life Si 6: , Pennimpede T, Cmeron DA, MLen GA, Li H, Au-Aed S, Petkovih M. The role of CYP26 enzymes in defining pproprite retinoi id exposure during emryogenesis. Birth Defets Res A Clin Mol Tertol 88: , Rosles FJ, Ritter SJ, Zolfghri R, Smith JE, Ross AC. Effets of ute inflmmtion on plsm retinol, retinol-inding protein, nd its mrna in the liver nd kidneys of vitmin A-suffiient rts. J Lipid Res 37: , Ross AC, Cifelli CJ, Zolfghri R, Li NQ. Multiple ytohrome P- genes re onomitntly regulted y vitmin A under stedy-stte onditions nd y retinoi id during hepti first-pss metolism. Physiol Genomis 3: 7 67, Ross AC, Zolfghri R. Cytohrome Ps in the regultion of ellulr retinoi id metolism. Annu Rev Nutr 31: 6 87, Ross AC, Zolfghri R. Regultion of hepti retinol metolism: perspetives from studies on vitmin A sttus. J Nutr 13: 269S 27S, Ruiz FX, Porté S, Gllego O, Moro A, Ardèvol A, Del Río-Espínol A, Rovir C, Frrés J, Prés X. Retinldehyde is sustrte for humn ldo-keto redutses of the 1C sufmily. Biohem J : 33 3, Ruiz FX, Porté S, Prés X, Frrés J. Biologil role of ldo-keto redutses in retinoi id iosynthesis nd signling. Front Phrmol 3: 217, Soprno DR, Blner WS. Plsm retinol-inding protein. In: The Retinoids: Biology, Chemistry nd Mediine, edited y Sporn MB, Roerts AB, Goodmn DS. New York: Rven, 199, p Sprrow JR, Wu Y, Kim CY, Zhou J. Phospholipid meets ll-trnsretinl: the mking of RPE isretinoids. J Lipid Res 1: , St CA, Ceder R, Roerg K, Grfström RC, Höög JO. Serum-responsive expression of ronyl-metolizing enzymes in norml nd trnsformed humn ul kertinoytes. Cell Mol Life Si 6: , 28.