| The incidence of central precocious puberty（CPP）is increasing year by year worldwide.CPP can accelerate the growth of bone age and impaired the growth potential,resulting in adult short stature.Growth hormone（GH）alone or GH+gonadotropin-releasing hormone analogs（Gn RHa）had a limited effect on height improvement in children with relatively older bone age and short stature.Aromatase inhibitors（AI）combined with GH are often tried to improve the height of relatively older bone age short boys,but relatively older bone age short girls lack safe and effective drugs and methods to inhibit bone age progression.Growth plate is the target organ for the longitudinal growth of the bone,and its premature closure caused by estrogen is one of the main causes of short stature in children with precocious puberty.Tamoxifen（TAM）is a non-sterol antiestrogen drug and is commonly used clinically to treat peripheral precocious puberty in girls caused by Mc Cunne-Albright syndrome（MAS）.In theory,as an estrogen antagonist,TAM may delay the acceleration of bone age caused by estrogen and improve the height of relatively older bone age short stature girls.However,there is a lack of relevant studies and data,and the specific mechanism of estrogen promoting growth plate closure is unknown.The previous view is believed that after apoptosis of the hypertrophy chondrocytes in the growth plate,osteoblasts invade cartilage with blood vessels,and then complete the process of osteogenesis and long bone growth.Recent findings suggest that within cartilage,chondrocytes can directly transdifferentiate into osteoblasts,suggesting that cartilage formation and osteogenesis may be a continuous biological process.However,whether estrogen directly promotes chondro-cytes trans-differentiation into osteoblasts and subsequently promotes growth plate closure remains unknown.In this study,we observed the effectiveness and safety of tamoxifen in a girl with relatively older bone age,and cultured human articular chondrocytes to explore the treatment method of short girls with relatively older bone age,and the effects and mechanisms of estrogen on the trans-differentiation of chondrocytes into osteoblasts.The study is divided into three parts:1)To observe the efficacy and safety of tamoxifen in a girl with relatively older bone age and short stature.2)To observe whether different concentrations of estrogen promote the trans-differentiation of chondrocytes into osteoblasts;3)To screen out the signaling molecules involved in chondrocyte trans-differen-tiation and their regulation mechanism;To go further and clarify the downstream signaling molecules involved in the signal pathway in order to provide a target and basis for the treatment of short stature girls caused by premature closure of growth plate.Part One Efficacy and safety of tamoxifen in a girl with relatively older bone age and short statureObjective:To observe the efficacy and safety of tamoxifen in a girl with relatively older bone age and short stature.Methods:A case of relatively older bone age short girl was selected:first visit at 11.0 years old,bone age 12.0 years（GP method）,height 138.1 cm（height for bone age-2SD）,BP method predicted adult height（PAH）149.8cm,breast TannerⅢstage,pubic hair Tanner II stage,without menarche.Tamoxifen（10 mg orally twice daily）was applied for six months.Height,growth velocity,bone age,PAH and adverse reactions were observed before and after treatment.Results:1.Baseline condition of the child before treatment1)General condition of the childThe girl was 11.0 years old at the first visit and she complained of her poor height increasing after treated with leuprorelin acetate for more than 1year.More than 1 year ago,the child was diagnosed with"early development"due to breast development,and leuprorelin acetate was prescribed（3.75 mg/4weeks,subcutaneous injection）.Her height increased slowly in the past half a year,and the growth velocity was about 4.1 cm/year.2)There was noting special in her birth,growth,family and past history.3)Physical examination:Height:138.1 cm（height for bone age-2SD）,weight:47.0 kg,well-symmetrical,no special face and rash,no abnormalities in lung,abdomen,nervous system,spine and limbs.Bilateral breast stage Tanner III,pubic hair stage Tanner II.4)Auxiliary examination:No abnormalities in routine blood,urine and stool,biochemistry,thyroid function,insulin-like growth factor-1（IGF-1）.Follicule-stimulating hormone 5.22 IU/L,luteinizing hormone 1.32 IU/L,estradiol 38.0pg/ml,uteroovarian ultrasound:uterine size 2.75 cm×2.58 cm×1.46 cm,endometrial thickness was about 0.33 cm,left ovary 2.21 cm×1.57cm×1.63 cm;right ovary 2.34 cm×1.79 cm×1.39 cm,there were several follicles larger than 4 mm.Chromosome 46,XX.Bone age（GP method）:12.0years old.5)PAH 149.8 cm.2.Conditions of the child after treatment1)Age:11.5 years old,no discomfort.Physical examination:Height:143.7 cm,weight:48.5 kg.There were more skin hair in the limbs and abdo-men,and no other abnormalities were found.Bilateral breast stage Tanner III and pubic hair stage Tanner II.2)Auxiliary examination:No abnormalities were found in routine blood,urine and stool,biochemistry,thyroid function,and IGF-1 test.Follicule-stimulating hormone 4.89 IU/L,luteinizing hormone 2.13 IU/L,estradiol40.0 pg/ml,uteroovarian ultrasound:uterine size 2.90 cm×2.59 cm×1.50 cm,endometrial thickness about 0.35cm,left ovary 2.35 cm×1.68 cm×1.72 cm;right ovary 2.56 cm×1.79 cm×1.41 cm,several follicles larger than 4mm.Bone age（GP method）:12.3 years old.3)Growth velocity was 11.2 cm/years,PAH 154.2 cm.The bone age increased 0.3 years and PAH increased 4.4 cm after treatment.Summary:1.Tamoxifen may be effective in delaying bone age and improving height in the child.2.Except for polyhairy,no other adverse reactions were seen in short-term tamoxifen application.Part Two Effect of 17β-estradiol on trans-differentiation of chondrocytesObjective:To study the effect of different concentrations of estrogen on chondrocyte differentiation.Methods:Human articular chondrocytes（CP-H096）were divided into control group and groups treated with different concentrations of 17β-estradiol（17β-E2）,and the concentrations of 17β-E2 were 10-8M/L,10-7M/L,10-6M/L and 10-5M/L respectively,and then were cultured for 7 days.Osteo-genesis was determined by alizarin red staining and alkaline phosphatase（ALP）activity measurement.In addition,blocked the estrogen receptor（ER）pathway using specific short hairpin RNAs（sh RNA）and tamoxifen（TAM）,and then compared the effect of E2 on ALP,osteocalcin（OCN）,Runt-related transcrip-tion factor 2（Runx 2）m RNA expression level and ALP activity before and after blocking using quantitative reverse transcription PCR（RT-q PCR）and ALP activity measurement.Results:1.Effect of different concentrations of 17β-E2 on osteogenesis indicators1)Comparison of mineralization results of different 17β-E2 concen-tration groupsThe results of alizarin red staining showed that the mineralization were150%±12%,210%±21%,280%±21%and 239%±18%in the 10-8M/L,10-7M/L,10-6M/L,and 10-5M/L 17β-E2 treated groups,respectively.The mineralization were significantly statistical different in 17β-E2 treated groups compared with the control group（P<0.01,P<0.001,P<0.001,and P<0.001,respectively）.Within a certain range,with the increase of E2 concentration,alizarin red staining gradually deepened,indicating that the mineralization and osteogenic conversion increased,and the mineralization reached the maximum value in the 10-6M/L group.2)Comparison of ALP activity in different 17β-E2 concentration groupsALP activity in the 10-8M/L,10-7M/L,10-6M/L,and 10-5M/L 17β-E2treated groups were 120%±12%,160%±21%,200±15%%,and 180±13%,respectively.ALP activity were increased in different 17β-E2 concentration treated groups compared with the control group（P<0.05,P<0.05,P<0.01,and P<0.01,respectively）,and ALP activity was highest in the 10-6M/L group.3)Comparison of the osteoblast marker m RNA in different 17β-E2concentration treated groupsALP m RNA expression level of control,10-8M/L,10-7M/L,10-6M/L,and 10-5M/L 17β-E2 treated groups were 1.06±0.08,1.26±0.08,1.56±0.08,1.76±0.10,and 1.59±0.06,respectively.Compared with the control group,The ALP m RNA expression level increased in different 17β-E2 concentration treated groups（P<0.05,P<0.05,P<0.01,and P<0.05,respectively）;The OCN m RNA expression level of each group were 1.01±0.10,1.19±0.05,1.35±0.07,1.66±0.06,and 1.49±0.08,respectively.Compared with the control group,OCN m RNA expression level in the 10-7M/L,10-6M/L,and 10-5M/L 17β-E2treated groups increased（P<0.05,P<0.01,P<0.05,respectively）;The Runx2m RNA expression level of each group were 1.00±0.09,1.53±0.06,1.86±0.09,2.10±0.06,and 1.76±0.06,respectively.Compared with the control group,the Runx2 m RNA expression level increased in different 17β-E2 concentration treated groups（P<0.05,P<0.01,P<0.01,and P<0.05,respectively）;ALP,OCN,and Runx2 m RNA expression level in the 10-6M/L 17β-E2 treated group were the highest among all groups.2.The effect of ER knockdown on osteogenesis1)Verification of ER knockdownThe ER m RNA expression level of the blank vector group（p LKO）was set to 1.00,and the ER m RNA expression level of sh ERα-1#,sh ERα-2#,sh ERβ-1#and sh ERβ-2#groups were 0.55±0.08,0.48±0.06,0.038±0.05,and0.66±0.09,respectively.Compared with the p LKO group,the ER m RNA expression level of each knockdown group decreased significantly（P<0.01,P<0.01,P<0.01,and P<0.05,respectively）.Western blot results showed that compared with the p LKO group,the ER protein expression in each knock-down group was also significantly reduced（P<0.01,P<0.01,P<0.01,and P<0.05,respectively）,indicating the success of ER knockdown.2)ALP and osteogenic marker detection after sh ER transfection of chon-drocytesALP activity was set to 100%in Con+p LKO group.After 10-6M/L E2treatment,ALP activity were 163%±12%,123%±9%,116%±10%,and 95%±9%in E2+p LKO,E2+sh ERα,E2+sh ERβand E2+sh ERα+sh ERβgroup,respec-tively.ALP activity was increased in the E2+p LKO,E2+sh ERα,and E2+sh ERβgroups compared to the Con+p LKO group（P<0.001,P<0.01,P<0.05,respectively）.ALP activity was significantly lower in E2+sh ERαand E2+sh ERβgroup compared to E2+p LKO group（both P<0.05）,and further decreased in E2+sh ER+sh ERβgroup compared to E2+sh ERαand E2+sh ERβgroup（both P<0.05）.ALP,OCN and Runx2 m RNA expression level in the Con+p LKO group was set to 1.00.After E2 treatment,the ALP m RNA expression level of each group was 1.69±0.08,1.34±0.10,1.23±0.09,and1.03±0.06,respectively.ALP m RNA expression level in E2+p LKO,E2+sh ERαand E2+sh ERβgroup increased compared to the Con+p LKO group（P<0.01,P<0.05,and P<0.05,respectively）;E2+sh ERαand E2+sh ERβgroup had decreased ALP m RNA expression level compared to E2+p LKO group（both P<0.05）,and in the E2+sh ERα+sh ERβgroup the ALP m RNA expression level was further decreased compared to the E2+sh ERαand E2+sh ERβgroup（both P<0.05）,consistent with the trend of ALP activity.After E2 treatment,the OCN m RNA expression level of each group was1.39±0.08,1.14±0.10,1.13±0.09,and 1.00±0.06,respectively,and was higher in the E2+p LKO group than Con+p LKO,E2+sh ERα,E2+sh ERβand E2+sh ERα+sh ERβgroup（P<0.01,P<0.05,P<0.05,and P<0.01,respec-tively）.After E2 treatment,the Runx2 m RNA expression level of each group was 1.89±0.08,1.54±0.10,1.43±0.09,and 1.13±0.06,respectively,and increased in the E2+p LKO,E2+sh ERαand E2+sh ERβgroup compared to the Con+p LKO group（P<0.001,P<0.01,and P<0.01,respectively）;E2+sh ERαand E2+sh ERβgroup had decreased Runx2 m RNA expression level compared to E2+p LKO group（both P<0.05）,and Runx2 m RNA expression level was further decreased in E2+sh ERα+sh ERβgroup compared to the E2+sh ERαand E2+sh ERβgroup（both P<0.05）.The above results indicated that the decrease in ERαor ERβled to a decrease of E2-induced osteogenesis,which is more pronounced when both ERαand ERβdecreased.3)Effect of tamoxifen on E2 promoting trans-differentiationThe ALP activity in the 10-6M/L E2-treated group（E2+Sham group）was163%±12%,which was significant increased than the control group（Con+Sham group）（P<0.001）;The ALP activity in the E2+1μM TAM group（E2+1μM group）was 113%±9%and was increased compared to the Con+Sham group（P<0.05）,but decreased compared to the E2+Sham group（P<0.05）;The activity of ALP in E2+5μM TAM group（E2+5μM group）was 98%±10%,and was decreased compared to the E2+Sham and the E2+1μM group（both P<0.05）;ALP,OCN,and Runx2 m RNA expression level in the E2+5μM group were 1.18±0.11,1.13±0.10 and 1.21±0.11,respectively,and were lower than E2+Sham group（in which the ALP,OCN,Runx2 m RNA expres-sion level were 1.72±0.08,1.46±0.09,and 1.89±0.10,respectively）（all P<0.05）.The results showed that 5μM TAM significantly inhibited the promoting effect of E2 on trans-differentiation.Summary:Within a certain range,high concentrations 17β-E2 promoted the trans-differentiation of chondrocytes in to osteoblasts via ERα/β.Part Three Signaling molecules involved in estrogen-promoting chondrocyte trans-differentiation and their regulatory mechanismsObjective:To screen out signaling molecules involved in the regulation of osteogenesis by 17β-estradiol/ER pathway and investigate their regulatory mechanisms.Methods:The chondrocytes were divided into blank vector Con+p LKO,E2+p LKO and E2+sh ERβgroups,and the E2 treatment concentration was 10-6M/L in the latter two groups.RT-q PCR detected the m RNA expression level of H subfamily of potassium voltage-gated channel 1（KCNH1）,phosphate regula-ting endopeptidase on the X chromosome（PHEX）,protein kinase c AMP-activated catal-ytic subunit alpha（PRKACA）,sortilin（SORT1）,insulin-like growth factor 2（IGF2）,dentin matrix acidic phosphoprotein 1（DMP1）,activating transcri-ption factor 5（ATF5）,bone morphogenetic protein 3（BMP3）,F-box only protein 5（FBXO5）,5-prime,3-prime-exori-bonu-clease1（XRN1）,zinc finger and homeodomain protein 3（ZHX3）and zinc finger protein 354C（ZNF354C）to screen out signaling molecules regulated by 17β-E2/ER pathway.Chondrocytes were divided into control group and groups treated with different concentrations of E2(10-8M/L,10-7M/L,10-6M/L,and 10-5M/L,respec-tively),and the m RNA and protein expression levels of each group were detected by RT-q PCR and Western blot;The chondrocytes were then divided into control group,TAM treated group（TAM concentration was 5μM）and TAM+E2 treated group(TAM concen-tration was 5μM and E2 concentration was 10-6M/L),and the protein expres-sion level of each group was detected.JASPAR database was used to predict the binding sites of ER and signaling molecular transcription factors,and chromatin immunoprecipitation（Ch IP-PCR）was used to confirm that;At last,the chondrocytes were divided into Con+p LKO group,E2+p LKO group,E2+sh ERαgroup,E2+sh ERβgroup and E2+sh ERα+sh ERβgroup.The E2treatment concentration in each group was 10-6M/L.The m RNA expression level and protein of participation signal molecules were detected by RT-q PCR and dual immunofluorescence staining respectively.Chondrocytes were divi-ded into p KLO（blank vector）and sh DMP1（knockdown DMP1）group.Protein kinase b（AKT）,phosphorylated protein kinase B（p-ATK）,extra-cellular regulated protein kinases（ERK）,phosphorylated extracellular regula-tory protein kinase（p-ERK）,glycogen synthase kinase 3β（GSK-3β）,phos-phoryl-glycogen synthase kinase 3β（p-GSK-3β）andβ-catenin of the two groups were detected using Western blot to screening out the signaling molecules downstream of E2/ER-DMP1;The chondrocytes were then divided into p KLO+Con,sh DMP1+Con and sh DMP1+E2 group,RT-q PCR was used for ALP,OCN,Runx2 andβ-catenin m RNA expression level;Chondrocytes were then divided into p KLO,sh DMP1,shβ-catenin（knock-down ofβ-catenin）and sh DMP1+shβ-catenin（simultaneous knockdown of DMP1 andβ-catenin）group,and the cell mineralization of each group was observed by alizarin red staining.Results:1.Signaling molecules involved in the 17β-estradiol/ER pathway1)DMP 1 is regulated by the 17β-estradiol/ER pathwayAmong the m RNA expression level of KCNH1,PHEX,PRKACA,SORT1,IGF2,DMP1,ATF5,BMP3,FBXO5,XRN1,ZHX3 and ZNF354C,the m RNA of DMP1,ATF5 and FBXO5 in the E2+p LKO group were statistical different compared to the Con+p LKO group（all P<0.05）;Only DMP1 m RNA expression level in the E2+sh ERβgroup had statistical difference compared to the E2+p LKO group（P<0.05）;After the E2 treatment,DMP1 m RNA expression level downregulated compared to the Con+p LKO group;After the knockdown of ERβ,DMP1 m RNA expression level upregulated compared to the E2+p LKO group,which suggested that DMP1 was regulated by the17β-estradiol/ER pathway.2)Comparison of DMP1 m RNA and protein expression level after treatment with different concentrations of E2Compared to the control group,both the DMP1 m RNA（P<0.01,P<0.001,and P<0.001,respectively）and protein（P<0.01,P<0.001,and P<0.001,respectively）expression level in the 10-7M/L,10-6M/L and 10-5M/L E2 treated groups decreased,which indicated the inhibitory effect of E2 on DMP1.3)Effect of TAM on the DMP1 proteinDMP1 protein increased after 5μM TAM treatment compared to control group（P<0.001）,while the TAM+E2 group showed less DMP1 protein than the TAM group（P<0.001）,which suggested that TAM blocks ER,attenuates the inhibitory effect of E2 on DMP1,and that E2 inhibits DMP1 through ER.2.E2/ER regulates DMP1 protein expression through transcription and promotes chondrocyte trans-differentiation1)Determination of the binding sites between DMP1 and ERWe predicted the binding sites for ER in the 2-kb 5′-promoter region of DMP1 using the JASPAR database.The results suggested that there might be one ERαbinding site and three ERβbinding sites in this region.Ch IP-PCR experiments indicated that the main regions of DMP1 binding to ERαand ERβwere between-1 and-182 bp（P<0.05 and P<0.001,respectively）.In addition,ERβhad binding sites in the-651 to-873 region upstream of the transcription start site within the DMP1 promoter（P<0.05）.2)Effect of ER knockdown on DMP1 m RNA and osteogenic trans-differentiation of chondrocytesCompared to Con+p LKO group,DMP1 m RNA expression level was significantly lower in E2+p LKO group（P<0.01）,while E2+sh ERα,E2+sh ERβand E2+sh ERα+sh ERβgroup had increased DMP1 m RNA expres-sion level compared to E2+p LKO group（P<0.05,P<0.05,and P<0.01,respec-tively）,which indicated that knockdown of ER was able to reverse the downregulation of DMP1 due to E2.Double immunofluorescence staining showed that DMP1 was downregulated in E2+p LKO,E2+sh ERαand E2+sh ERβgroup compared to Con+p LKO group（P<0.01,P<0.05,and P<0.05,respectively）,while DMP1 was increased in E2+sh ERα+sh ERβgroup com-pared to E2+sh ERαand E2+sh ERβgroup（both P<0.05）;The level of Runx2in each group was quite opposite to that of DMP1（when E2+p LKO,E2+sh ERαand E2+sh ERβgroup were compared to the Con+p LKO group,P<0.001,P<0.01,and P<0.01,respectively;and E2+sh ERα+sh ERβgroup was compared to the E2+sh ERαand E2+sh ERβgroup,both P<0.05）.The results showed that E2 inhibited the DMP1 levels and promoted the trans-differentiation of chondrocytes into osteoblasts,and the knockdown of ER could reverse the above effects.3.Detection of signaling molecule expression after DMP1 knockdownAfter DMP1 knockdown,protein expression of p-GSK-3β and β-catenin increased（both P<0.05）but no significant changes were found in AKT and ERK（both P>0.05）compared to p KLO group.The result indicated that p-GSK-3β/β-catenin was regulated by DMP1.4.β-catenin participates in the E2/ER-DMP1 pathway in regulating the trans-differentiation of chondrocytes into osteoblastsRT-qPCR showed that after DMP1 knockdown,β-catenin m RNA expression level increased in sh DMP1+Con group compared to p KLO+Con group,and the changes of ALP,OCN and Runx2 m RNA expression level were consistent withβ-catenin m RNA（all P<0.05）.Alizarin red staining showed that mineralization increased in the sh DMP1+Con group but decreased in the shβ-catenin group compared to the p KLO+Con group（both P<0.05）;the mineralization in the sh DMP1+shβ-catenin group was lower than sh DMP1group and higher than the shβ-catenin group（both P<0.05）.The above results showed that DMP1 knockdown increased the levels ofβ-catenin and osteo-genic markers,and simultaneous knockdown of DMP1 andβ-catenin could reverse the inhibitory effect of mineralization byβ-catenin knockdown alone,indicating that E2/ER promoted chondrocyte trans-differentiation by upregu-lating GSK-3β/β-catenin through inhibiting DMP1.Summary:1.E2/ER promotes the trans-differentiation of chondrocytes into osteo-blasts by inhibiting DMP1 transcription.2.Inhibition of DMP1 upregulated GSK-3β/β-catenin and promoted chondrocyte trans-differentiation.Conclusions:1.Tamoxifen may be a potential drug to delay bone age and improve the height of short girls with relatively older bone age.2.Within a certain range,high concentrations of E2 inhibited DMP1transcription through ERα/β,upregulated GSK-3β/β-catenin,and finally promoted the trans-differentiation of chondrocyte into osteoblasts. |
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