Osteogenic Oxysterol, 20(S )-Hydroxycholesterol,Induces Notch Target Gene Expression in Bone MarrowStromal CellsWoo-KyunKim,1VicenteMeliton,1SotiriosTetradis,2GerryWeinmaster ,3TheodoreJHahn,4MarcCarlson,5StanleyFNelson,5andFarhadParhami11Department of Medicine, UCLA School of Medicine, Los Angeles, CA, USA2UCLA School of Dentistry, Los Angeles, CA, USA3Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA, USA4VA Greater Los Angeles Healthcare System and Geriatric Research, Education, and Clinical Center, Los Angeles, CA, USA5Department of Human Genetics, UCLA School of Medicine, Los Angeles, CA, USAABSTRACTWepreviouslyreportedthatspecificoxysterolsstimulateosteogenicdifferentiationofpluripotentbonemarrowstromalcells(MSCs)throughactivationof hedgehog(Hh) signalingandmay serveas potential futuretherapies for interventioninosteopeniaandosteoporosis. Inthisstudywereportthattheosteogenicoxysterol 20(S)-hydroxycholesterol (20S) inducestheexpressionof genesassociatedwithNotchsignaling. UsingM2-10B4(M2)MSCs, wefoundthat20SsignificantlyinducedHES-1, HEY-1, andHEY-2mRNAexpression compared with untreated cells, with maximal induction after 48 hours, whereas the nonosteogenic oxysterols did not. Similarobservations were made when M2 cells were treated with sonic hedgehog (Shh), and the specific Hh pathway inhibitor cyclopamineblocked20S-inducedNotchtargetgeneexpression. 20Sdidnotinduce NotchtargetgenesinSmo/mouseembryonicfibroblasts,further confirming the role of Hh signaling in 20S-induced expression of Notch target genes. Despite the inability of liver X-receptor (LXR)syntheticligandTO901317toinduceNotchtargetgenesinM2cells, LXRknockdownstudiesusingsiRNAshowedinhibitionof20S-induced HEY-1 but not HES-1 expression, suggesting the partial role of LXR signaling in MSC responses to 20S. Moreover, 20S-inducedNotchtargetgeneexpressionwasindependentofcanonical Notchsignalingbecauseneither20SnorShhinducedCBF1luciferasereporter activity or NICD protein accumulation in the nucleus, which are hallmarks of canonical Notch signaling activation. Finally, HES-1and HEY-1 siRNA transfection significantly inhibited 20S-induced osteogenic genes, suggesting that the pro-osteogenic effects of 20S areregulatedinpartbyHES-1andHEY-1. 2010AmericanSocietyforBoneandMineral Research.KEYWORDS: OXYSTEROL; MESENCHYMALSTEMCELLS; NOTCH; HEDGEHOG; OSTEOGENESISIntroductionTheNotchsignalingpathwayisanevolutionarilyconservedintercellular signaling mechanismthat plays a prominent rolein cell proliferation, differentiation, and survival.(1,2)Thecanonical Notchsignalingpathway is activatedwhenNotchreceptors (Notch-1, -2, -3, and -4) interact with ligands [Jagged-1and -2 and Delta-like (Dll-1, -3, and -4)] on adjacent cells,triggering proteolytic cleavage of the receptor by the preseniling-secretase complex.(1,2)This releases the Notchintracellulardomain (NICD), which translocates to the nucleus and binds theCBF-1 DNA-binding protein, thereby inducing the expression ofNotchtarget genes, includingtheisoforms of HES(HES-1, -3,and -5) and HEY (HEY-1, -2, and -3).(3)These Notch target genesareinvolvedinvariousbiologicprocesses, includingangiogen-esis, osteogenesis, adipogenesis, myogenesis, somatogenesis,andneurogenesis.(49)Regulationof Notchsignalingpathwayand target gene expression is important in embryonic andpostembryonic development and tissue homeostasis.(1,1012)However, it remains controversial as to whether Notch signalingacts as a positive or negative regulator of osteogenicdifferentiation in osteoblast progenitor cells and bone formation.For example, Dll-3- or presenilin-1-deficient mice exhibit severeskeletal defects,(8,13,14)and overexpression of Notch-1, HES-1, orORIGINALARTICLEJReceivedinoriginal formMarch9, 2009; revisedformSeptember21, 2009; acceptedOctober15, 2009. PublishedonlineOctober17, 2009.Address correspondence to: Farhad Parhami, PhD, David Geffen School of Medicine at UCLA Center for the Health Sciences, A2-237, 10833 Le Conte Avenue, LosAngeles, CA90095, USA. E-mail: fparhami@mednet.ucla.eduJournal ofBoneandMineral Research, Vol. 25, No. 4, April 2010, pp782795DOI: 10.1359/jbmr.0910242010AmericanSocietyforBoneandMineral Research782HEY-1 enhances osteogenic differentiation of MSCs(1517)in partthroughpositive regulationof andcooperation withRunx2,suggesting that Notch signaling may play positive roles in boneformation. On the other hand, presenilin-2 null mice have greatlyincreased trabecularbone mass, and HES orHEYproteinswereshowntoinhibitRunx2transcriptional activityinCHOandST2cells, suggesting the negative role of Notch signaling inosteogenesis.(18)However, it also has been suggested thatHES and/or HEY expression induced by Notch signaling may beimportant inregulatingbonedensityduringagingbymain-tainingasufficient pool of bonemarrowprogenitor cells forosteogenesis.(18)Therefore, further examinationof theroleofNotch signaling in regulating osteogenesis and bone formationis required, and it is likely that the differences in the reports citedearliermaybeduetodifferencesinthespecificexperimentalmodels used in studying the role of Notch signaling inosteogenesis.Inadditiontocanonical Notchsignaling, theexpressionofNotchtarget genes is regulatedbygrowthfactors, includingtransforming drowth factor b(TGF-b), bone morphogeneticprotein(BMP), vascular endothelial growthfactor (VEGF), andsonic hedgehog (Shh).(17,1921)TGF-b induces HEY-1 and Jagged-1inepithelial cellsfrommammarygland, kidneytubules, andepidermis,(19)and BMP-9 induces HEY-1 expression in C3H10T1/2cells.(17)Also, ShhandVEGFinduceNotch-5andHES-1mRNAexpressioninvariouscells, includingC3H10T1/2cells, MNS70neural cells, andgranuleneuronprecursors.(2022)Moreover, ithas been suggested that regulation of HES-1 expression by c-Junkinase signalingandHedgehogsignaling may be mediatedthroughtheactivationof noncanonical Notchsignalingpath-ways.(2224)Hence the molecular mechanisms by which growthand differentiation factors activate the Notch signaling pathwayand induce the expression of Notch target genes require furtherelucidation.Oxysterols, a large family of 27-carbon oxygenated products ofcholesterol present in the circulation and in human and animaltissues,(25)are involved in various biologic and pathologicprocesses, includingcholesterol efflux, lipoproteinmetabolism,cell differentiation, atherosclerosis, and apoptosis.(2629)We havedemonstratedpreviouslythat specificoxysterolsstimulatetheosteogenicdifferentiationof pluripotentMSCs andinhibit theiradipogenicdifferentiationthroughtheactivationofHedgehogsignaling in vitro(3033)and enhance bone healing in rat critical-sized calvarial defects in vivo.(34)Here, we report that osteogenicoxysterols are novel activators of expression of the Notch targetgenes HES-1, HEY-1, and HEY-2 in MSCs. Moreover, the inductionof Notch target gene expression by 20S is not mediated by thecanonical Notchsignalingpathwaybut mainlybyHedgehogsignalingandinpart byLXRsignaling, andHES-1andHEY-1induction appears necessary for maximal induction of osteogen-esisby20S.MaterialsandMethodsCell cultureandreagentsM2-10B4 (M2) pluripotent mouse marrow stromal cells andSmo/mouse embryonic fibroblasts (MEFs) were maintained asdescribedpreviously.(31,32,35,36)Cell treatment wasperformedindifferentiation medium containing 5% fetal bovine serum(FBS), 50mg/mL ascorbate, and 3 mM b-glycerophosphate.Oxysterols were purchasedfromSigma-Aldrich, Co. (St. Louis,MO, USA); N-[N-(3,5-difluorophenacetyl-L-alanyl)] S-phenylglycinet-butylester(DAPT)andcyclopaminewerefromCalbiochem(LaJolla, CA, USA), andrecombinantmouseShhN-terminalpeptideandJagged-1 werefromR&DSystems (Minneapolis, MN, USA).Quantitativereverse-transcriptasepolymerasechainreaction(qRT-PCR)Total RNA was extracted with an RNA isolation kit fromStratagene(LaJolla, CA, USA)accordingtothemanufacturersinstructions. RNAwasDNasetreatedusingaDNA-freekitfromAmbion (Austin, TX, USA). Then 3 mg of RNA was reverse-transcribed using reverse transcriptase from Stratagene (La Jolla,CA, USA)tomake single-strandedcDNA. The cDNAsthenweremixed with Qi SYBR Green Supermix (Bio-Rad, Hercules, CA, USA)for qRT-PCR assay using a Bio-Rad I-cycler IQ quantitativethermocycler. All PCR samples were prepared in triplicate wells ina 96 well plate. After 40 cycles of PCR, melt curves were examinedtoensureprimer specificity. Foldchangesingeneexpressionwere calculatedusing theDDCtmethod andnormalized totheexpression of the housekeeping gene GAPDH. Primers usedwere as follows: HES-1: 50-TACCCCAGCCAGTGTCAACA-30and50-CCATGATAGGCTTTGATGACTTTCT-30 (37); HEY-1: 50-TGAGCTGA-GAAGGCTGGTAC-30and 50-ACCCCAAACTCCGATAGTCC-50 (38);HEY-2: 5V0-TGAGAAGACTAGTGCCAACAGC-30and50-TGGGCAT-CAAAGTAGCCTTTA-30(38); Jagged-1: 50-TGGTTGGCTGGGAAATT-GA-30and50-TGGACACCAGGGCACATTC-30 (39); Delta-1: 50-CAC-TATGGACAGTTGCTTTGAAGAGT-30and50-TGGCTCATAGTAATC-CAAGATAGACG-50 (40); Notch-1: 50-GGATCACATGGACCGATTGC-30and 50-ATCCAAAAGCCGCACGATAT-30 (39); Notch-2: 50-CCCCT-TGCCCTCTATGTACCA-30and 50-GGTAGGTGGGAAAGCCACACT-30 (39); ALP: 50-AAACCCAGAACACAAGCATTCC-30and50-TCCAC-CAGCAAGAAGAAGCC-30; ABCA1: 50-TGCCACTTTCCGAATAAAGC-30and 50-GGAGTTGGATAACGGAAGCA-30; BSP 50-ACGCCA-CACTTTCCACACTCTC-30and 50-TTCCTCTTCCTCTTCTTCTTCTTC-TTCC-30; and GAPDH: 50- ATGGACTGTGGTCATGAGCC-30and50-ATTGTCAGCAATGCATCCTG-30.CBF-1luciferaseassayM2cellsat 70%confluencyin24well platesweretransientlytransfected with CBF-1 luciferase reporter construct pTK-luciferase plasmid and pTK-Renilla-luciferase plasmid (Promega,Madison, WI, USA)usingFugene6TransfectionReagentsfromRoche (Indianapolis, IN, USA).(2)Twenty-four hours aftertransfection, thecellsweretreatedwithcontrol vehicleorNotchinteracellular domain (NICD) overexpression vector with or without5mM20Sand200ng/mLmouserecombinantShhfor24and48hours, andNotchactivationof CBF-1wasnormalizedtoRenillaluciferase activity. Transfection efficiency was monitored bycotransfecting with a plasmid expressing green fluorescent protein.Jagged-1, Notchintracellulardomain(NICD), andHES-1WesternblotFor Jagged-1 Western blot, M2 cells at confluence were treatedwith control vehicle (control), 5 mM20(S)-hydroxycholesterolOXYSTEROLINDUCESNOTCHTARGETGENEEXPRESSION JournalofBoneandMineralResearch 783(20S), and 200 ng/mL sonic hedgehog (Shh). After 48 or 72 hoursof treatment, whole-cell lysates were collected, and proteinconcentrations were determined using the Bio-Rad proteinassay. For NICD Western blot, M2 cells at 100% confluence weretreated with control vehicle (control) or 5 mM 20S or cultured on5 mg/mLimmobilizedJagged-1. After48and72hours, nuclearextractswerecollectedandprotein concentrationsdeterminedusingtheBio-Radproteinassay. ForWesternblottingofHES-1andb-actin, whole-celllysateswerecollectedafter72hoursofcontrol vehicleor 5 mM20Streatment inM2cellstransfectedwith either scramble control or HES-1 siRNA. The samplesweresubjectedtosodiumdodecylsulfatepolyacrylamidegelelectrophoresis (SDS-PAGE) andtransferredovernight ontoanitrocellulose membrane(AmershamBiosciences, Piscataway,NJ, USA). BlotsthenwereincubatedwithpolyclonalantibodiesagainstHES-1fromSantaCruzBiotechnology(SantaCruz, CA,Fig.1.20(S)-Hydroxycholesterol (20S) induces Notch signaling target genes HES-1, HEY-1, and HEY-2 in M2-10B4 bone marrowstromal cells. (AC) M2 cellswere treated at confluence with control vehicle or 5 mM20S, 7a-hydroxycholesterol (7-aHC), or 7-ketocholesterol (7-ketoC) for 48 hours. HES-1, HEY-1, andHEY-2 mRNA expression was measured by quantitative real-time PCR. (DF) M2 cells were treated at confluence with control vehicle or 5 mM20S for 24, 48,and 96 hours. HES-1, HEY-1, and HEY-2 mRNA expression was measured by quantitative real-time PCR. Fold changes in gene expression compared with thecontrol were calculated using the DDCt method and reported as the mean of triplicate determination SD (AC:p