Fatty Acid Synthase Expression In Periosteum Tissue Of Rat

Objective:estrogen plays a key role in pathogenesis of postmenopausal osteoporosis.Bone formation under periosteum is repressed by estrogen during adolescent phase,andenhanced after menopause,which compensates,at some extent,the decreased bonestrength induced by bone mass loss.Supplementation of estrogen could further increasebone formation under periosteum.We hypothesized that estrogen prevent fromosteoporosis through,at least in part,periosteum,its mechanism at gene level,however,is still far from clarification.We have constructed a subtracted cDNA library after estrogentreatment in previous studies,and the number of genes regulated by estrogen inperiosteal cells were screened.Based on these results,fatty acid synthase gene wasfound to be related to 17 8 estradiol.In this study,we have explored the possiblealternation during growth,and under the influence of estrogen,the tissue distribution of itsprotein,as well as relationship with bone histomorphometry and bone mass.Methods1.Screening and verificationCells were enzymatically isolated from the human periosteum by two-step sequentialdigestion with trypsin and type I collagenase and identified thereafter.Total RNA wasextracted from primary periosteal cells after 7 days incubation with estrogen,and thencDNA formed by reverse transcription.Specific primers were designed for the 22 genes,which repeated more than 10 times in sequencing results.The first screening procedurewas done with semi-quantitative PCR,and real-time PCR was introduced for theclarification.Further animal experiments was performed for the studies of fatty acidsynthase (FAS),the gene with most significantly different change of expression afterestrogen treatment.2.Animal experiments:this section is composed of three parts.(1) FAS expression at different development stages:10 Sprague-Dawley (SD) rats wereselected in each life group of 5 days,3 months,8 months and 16 months,respectively.(2) FAS expression before and after ovariectomy.30 rats of 6 months were randomized byweight into 3 equal groups.(3) FAS expression in different stages of estrous cycle.Pre-estrous,estrous,post-estrousand inter-estrous rats of 3 months identified by vaginal smear examination were classifiedas 4 equal groups,each composed of 10.BMD were measured before these rats werescarified. In all three parts,BMD and body composition and fat distribution were measured beforethese rats were scarified.Serum was collected for estrogen and progesterone levels bychemiluminescence assay,calvaria periosteum was collected for extracting total RNA andreverse transcription done to obtain cDNA.Semi-quantitative PCR was executed to detectFAS expression.Femur at left side was collected for bone histomorphometry;and rightfemur tissue with periosteum was used for FAS expression by immunohistochemistry.Results(1) 7 genes were screened and rectified by semi-quantitative PCR,which were:cytochrome P450,family 1,subfamily B,polypeptide 1 (CYP1B1);non_metastatic cells 4(NME4);cathepsin B (CTSB);beta₁,3_glucuronyltransferase 3 (glucuronosyltransferaseI) (B3GAT3);protein kinase N1 (PKN1);thrombospondin 1 (THBS1);fatty acid synthase,FAS.The latter three genes were further verified by real-time fluoresce quantitative PCR.(2) Alterations of FAS expression1 ) For rats in different development stagea.The sequence of FAS gene expression levels was as following:neonatal rats＞ratsaged 16 months＞rats aged 9 months (3.38±3.74,3.04±1.07,1.51±1.32 and 0.52±0.34,respectively),the 3 months rat had significant low level of the gene expression comparedwith neonatal and 16-month rats.b.Immunohistochemistry revealed positive periosteal FAS gene expression in all rat withdifferent development stages,mainly in fibrous layer while extraordinary strong periostealFAS gene expression in neonatal rats,particularly in the formation layer.2) For OVX,Sham and control groups:operation performed at 6-month,and specimencollected at 9-month.a.Serum levels of estradiol were:sham group＞control group＞OVX group (20.81±12.62pg/ml,17.64±4.35 pg/ml and 13.47±1.84/pg/ml).b.OVX group had significantly higher FAS gene expression than sham and control group(3.09±1.97,1.33±0.47,1.51±1.32),(p=0.0372).c.Immunohistochemistry revealed positive periosteal FAS gene expression in all groups,mainly in fibrous layer.3) For rats in different stages in estrous cycles:3-month rats used.a.Serum levels of estradiol were:pre-estrous rats＞estrous rats＞inter-estrous rats＞post-estrous rats (36.11±24.45pg/ml,24.49±19.46pg/ml,22.95±7.94pg/ml and14.78±2.49pg/ml,respectively),with significant difference between pre-estrous andpost-estrous rats (p= 0.0270).b.FAS gene expressed in periosteum were:post-estrous rats＞pre-estrous rats＞inter-estrous rats＞estrous rats (36.11±24.45,24.49±19.46,22.95±7.94 and 14.78±2.49,respectively),with significant difference between post-estrous rats and the other 3 groups (P=0.0001).c.Immunohistochemistry revealed positive periosteal FAS gene expression in all groups,mainly in fibrous layer.4) Bone histomorphometry results from 3-,9- and 16-month rats:FAS gene periostealexpression in 9- and 16 month rats had a negative correlation with parameters in bonehistomorphometry such as:trabecular area (r=-0.44018,p=0.0025),percentage oftrabecular area (r=-0.44018,p=0.0025),trabecular numbers (r=0.62069,p=0.0001.),butpositive with trabecular separation (r=?,p=.0.0001).5) Bone mineral density (BMD) results from 3-,9- and 16-month rats:FAS gene periostealexpression in 3- month rats had a negative correlation with spine BMD (r=-0.37122,p=0.0398) and total body BMD (r=-0.35998,p=0.0467).Conclusion(1) multi-gene expression is changed after estrogen treatment.(2) Estradiol inhibits FAS gene expression in human periosteal cells in vitro.(3) Estradiol inhibits FAS gene expression in periosteum of rat in vivo.(4) FAS gene expression in periosteum has negative correlation with BMD in 3-month ratsand no correlation with fatty content.(5) FAS gene expression in periosteum has negative correlation with bone mass andpositive correlation with trabecular deformity as well in 9- and 16-month rats.(6) Immunohistochemistry revealed extraordinary strong positive periosteal FAS geneexpression in the formation layer of neonatal rats,for the other adult rats,the gene has apositive expression in fibrous layer.(7) FAS gene is likely to be involved in bone metabolism under estrogen action.