1.Mechanism Of Rosiglitazone Regulating Proliferation And Differentiation Of The Cranial Osteoblast2.Isolfavones Regulate Estrogen Response Element And CAMP Response Element-mediated Transcription In Osteoblastic Cell Line

Osteoporosis (OP) is a common bone disease characterized by low bone mass,microarchitectural deterioration of bone tissue and increased risk of fracture. The mainclinical symptoms were pain and fracture of bone. OP is a worldwide health problem,which seriously disturbs quality of people’s health and life, with heavy economic andsocial burden resulted from disability, malformation and even death induced by OP. It’simportant to find ways to prevent and treat OP effectively.According to pathogeny, it can be divided into two groups: primary OP and secondaryOP. Post-menopause Osteoporosis is a typical of the primary OP, which has physiologicdegeneration along with the age growth. And the secondary OP is caused after otherdiseases or drug treatments. For example, the concern has been gradually improved aboutthe secondary OP induced after thiazolidinediones (TZDs) treatment of diabetes. Althoughthe reasons of OP are complicated, the direct cause is disorder of bone metabolism. Bonemetabolism continually keeps a dynamic balance with bone formation and bone absorptionduring one’s lifetime. These works are carried by the osteoblasts (OB) and osteoclasts(OC). If the function of osteoblasts was weaker than osteoclasts, the bone mass woulddecrease; and heavily decrease of bone mass would cause OP. As the main functional cell,osteoblasts play a key role in keeping balance of bone metabolism. And the proliferationand differentiation of OB can be regulated by multiple factors. For this, we chose twokinds of typical OP, TZD-induced OP and post-menopause OP. Our research is divided intotwo parts to study mechanisms of TZDs and soybean isoflavone regulate OB.The first part of our work is mainly about the TZD-induced OP. TZDs serve as anagonist for the peroxisome proliferator activated receptor gamma (PPARγ) to treatpatients with Type2Diabetes. And PPARγ is a subunit of PPAR, which belongs tonuclear receptor superfamily, a key treating target of metabolic diseases (such as diabetes,atherosclerosis). However, recent clinical trials show that long term TZDs treatment leadsto increased risk of fracture. Laboratory studies in animals have found that both PPARγexpression and endogenic ligand of PPAR γ increase can significantly inhibit OB’sfunction, stimulate OC’s activity and promote bone absorption. All of above suggest thatPPARγ play a moderating role in the bone metabolism. But the mechanisms are stillunclear. OBs are of mesenchymal lineage in bone marrow. Recent studies havedemonstrated that TZDs can enhance preferential differentiation of mesenchymal stem cells into adiopocyte at the expense of osteoblasts in the bone marrow. However,controversies exist in their effect on late stage osteoblast differentiation. Some studiesshowed that RSG strongly affect the early stage OBs differentiation, rather than late stages.In contrast, some papers claimed that TZDs inhibit late stage osteoblasts differentiation andproliferation. Therefore, the effect of TZDs on late stage osteoblast differentiation stillremains unclear. Meanwhile, recent research finds that Src-homology domain-2phosphatase1(SHP-1) may take part in the regulation of bone metabolism. It can decreasebone mass lost through inhibiting osteoclastic activity. But the effects on OBs remainunclear.In view of above, we use rosiglitazone (RSG) as the typical TZD and chose theprimary cultured calvarial osteoblasts from newborn mice as the model of late stageosteoblasts. This part of work mainly includes two aspects as follows.1. to clarify effectsof rosiglitazone on calvarial osteoblast’s differentiation;2. to clarify effects ofrosiglitazone on calvarial osteoblast’s proliferation.The second part of our works is mainly on the osteoblastic cell line MG-63. Thetraditional estrogen replacement therapy (ERT) is widely used in the treatmentof post-menopausal osteoporosis. However, there are some potentially dangerous such asbreast cancer, endometrial cancer and etc. al. recently, it is being paid great attention thatthe importance of phytoestrogen in the treatment of osteoporosis. Soybean isoflavone isone typical kind of phytoestrogen. It belongs to plant flavonoids, mainlyfrom Leguminosae Papilionoideae plant. It is a non-sterol material havingwidely nutrition value of health protection and therapy. Both animal studies and clinicaltrials show that soybean isoflavone has protection effects on bone, but mechanism is notclarified. Because its structure is similar to that of estrogen, Soybean isoflavone can bind toestrogen receptors leading transcriptional regulation. As we all known, ERs have twosubtypes: ER α and ER β. After binding with ERs, estrogen regulates target genetranscriptional activity through ERE; or through directly cross-talking with protein toregulate CRE transcriptional activity. Whether isoflavones has same effects as estrogen inthese two ways, there are different reports about it.So, using human osteosarcoma MG-63cell line, we precede this part of work with twoaspects as follows:1. Compare mechanisms of soybean isoflavone and estrogen throughestrogen classic pathway, which is ERE-dependent pathway;2. Compare mechanisms ofsoybean isoflavone and estrogen through non-estrogen classic pathway, which is CRE-dependent pathway.Results:Ⅰ. Rosiglitazone regulates proliferation and differentiation ofcalvarial osteoblasts1. Rosiglitazone inhibits differentiation of calvarial osteoblasts1). Primary cultured calvarial osteoblasts from newborn mice are used as the model oflate stage osteoblasts. For differentiation, ascorbic acid (50uM), and beta-glycerophosphate(10mM) were used to induce pre-osteoblast maturity (IM). After continuously treating cellswith rosiglitazone （10-9～10-6M）for10days, we found that RSG can inhibits fourosteogenic genes (ALP, Col1, OC and Runx2) mRNA expression in dose-dependent.2). Cultured with IM condition and treated with rosiglitazone （10-9～10-6M）for21days, cells were measured with alizarin red staining and alkaline phosphatase activity. Wefound that RSG dramatically inhibit calcium nodule formation and activity of alkalinephosphatase, which suggests RSG can suppress calvarial osteoblasts differentiation.3). It is well known that-catenin plays key role during osteoblasts maturation. As themost important factor of regulating Wnt signal pathway, the level of-catenin can beregulated by GSK3. When GSK3is activated, the level of-catenin will decreaseinduced by degradation arise, which inhibit the osteogenesis. In order to verify whetherGSK3pathway take part in the effects of RSG on osteogenesis, we use specificantagonists of GSK3(SB216763) co-act with RSG. We found that SB216763can reversethe inhibition of osteogenic genes (ALP, Col1, OC and Runx2) mRNA expression and-catenin protein expression caused by RSG.4). We also measured SB216763effects on calcium nodule formation and activity ofalkaline phosphatase. After co-treating with SB216763and RSG for21days, we foundSB216763can also reverse the inhibition of RSG. All above suggests RSG inhibitsdifferentiation of calvarial osteoblasts through activating GSK-3.5). To further clarify the mechanism of RSG inhibition effects, we treated cells withRSG (10-6M）and GW9662(10-6M), PPARG specific inhibitor. we found that RSGinhibition effects on OBs differentiation can be partly reversed. Furthermore, using siRNAinterference to knock down PPAR, the results are the same as GW9662. That suggestsRSG activate GSK3partly through PPAR. 6). There are some reports that RSG could play a role in OC through GPR40. So, wewant to verify whether RSG works through GPR40in OB. First, we use GW9508(10-5M)alone, the agonist of GPR40, and there are no effects on osteogenic genes mRNAexpression. Then, after using siRNA interference to knock down GPR40, we treated thecells with RSG. And it was also found that RSG inhibition effects be partly reversed. Wealso found that the lever of GPR40is high and PPAR is low without IM. However, afterIM, PPAR raise and GPR40decrease. When treated with RSG, both GPR40and PPAR have a rise with dose-dependent manner. All above suggest that GPR40remains low leverduring IM, RSG can stimulate GPR40protein expression which have parts of RSG effects.7). We also found mRNA and protein expression of SHP-1increased during inducedOB maturity in time-dependent. We treat cells with different dose of NSC87877, thespecific antagonists of SHP-1. NSC87877significantly inhibits the osteogenic gene mRNAexpression and protein expression of-catenin with dose-dependent. Furthermore, usingsiRNA interference to knock down SHP-1or over express catalyticallyinactive SHP-1mutant plasmid in calvarial osteoblasts, we found that osteogenicexpression gene is significantly inhibited. All above suggests SHP-1could stimulate theosteogenesis.8). when the cells were co-treated with SB216763(2*10-5M) and NSC87877,SB216763can reverse NSC87877inhibition effects on-catenin protein expression. Itsuggests SHP-1inhibit calvarial osteoblasts differentiation through activating GSK3.9). After treating with RSG (10-6M), we found that SHP-1mRNA expression aredecrease. It suggests that RSG can inhibit SHP-1mRNA expression in calvarialosteoblasts.2. Rosiglitazone inhibits proliferation of calvarial osteoblasts1). Culturing with IM condition and treated with rosiglitazone （10-9～10-6M）for5days, using MTT （3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide）, wefound that RSG inhibits proliferation of calvarial osteoblasts in dose-dependent.2). Cells were treated with SB216763(2*10-5M) and RSG (10-6M）. we found thatSB216763can reverse the inhibition of RSG. All above suggests RSG inhibits proliferationof calvarial osteoblasts also through activating GSK-3.3). To further clarify the mechanism of RSG inhibition effects, we treated cells withRSG (10-6M）and GW9662(10-6M), PPARG specific inhibitor. we found that RSGinhibition effects on OBs proliferation can not be changed. Furthermore, using siRNA interference to knock down PPAR, the results are the same as GW9662. That suggestsRSG inhibits OB’s proliferation through PPAR-independent way.4). To verify whether RSG inhibits proliferation through GPR40, we knock downGPR40. Then, RSG’s inhibition effects on proliferation were reversed. It suggests RSGinhibits proliferation of calvarial osteoblasts through GPR40.5). We also observe the SHP-1effects on OB’s proliferation. Using siRNA interferenceto knock down SHP-1, the proliferation is significantly decreased. It suggests that SHP-1have positive regulation on OB’s proliferation.Ⅱ. Isoflavones regulate estrogen response element and cAMPresponse element-mediated transcription in osteoblastic cell line1. Isoflavones regulate estrogen response element-mediatedtranscription1). First, we compare effects of estrogen and soybean isoflavone on the classicalestrogen pathway. Overexpressed ER and ER and treated with different dose of estrogenand isoflavone (daidzein, genistein, puerarin, kaempferol, quercetin), it was found thatestrogen (10-10～10-8M), daidzein and genistein （10-6M，10-7M） significantly stimulateERE report gene expression when ER overexpressed. The rest phytoestrogen have noeffects except kaempferol. And kaempferol has weak effect only in high dose（10-6M）.When ER was overexpressed, effects of kaempferol and quercetin are same as ER overexpressed. Estrogen (10-9～10-6M), daidzein and genistein significantly stimulateERE report gene expression. Effects of daidzein and genistein are stronger than ER overexpressed. However, puerarin inhibit ERE report gene transcriptional activity withdose-independent manner.2). Co-transfected with ERE report gene and ER or ER, treated with ER antagonistICI182,780and (selective estrogen regulate mediator, SERM）4-OH-tamoxifen, it wasfound that ICI182,780inhibits ERE report gene transcriptional activity in dose dependentmanner only ER overexpressed. But4-OH-tamoxifen effect is different through differentER subunit. When ER was overexpressed,4-OH-tamoxifen stimulates ERE report genetranscriptional activity in dose dependent manner. However, when ER was overexpressed,4-OH-tamoxifen inhibits gene transcriptional activity in dose independent manner.3). Overexpressed ER and treated with estrogen or phytoestrogen and ICI182,780or 4-OH-tamoxifen, it was found that effects of estrogen were totally inhibited, effects ofdaidzein and genistein were partly inhibited, but effects of kaempferol were no change.When ER was overexpressed, effects of estrogen, daidzein, genistein and kaempferol arealmost totally inhibited, but effects of puerarin are not change. Since puerarin has effectssame as antagonist of ER, effects of estrogen, daidzein, genistein and kaempferol can alsobe partly inhibited when they were treated with puerarin.2. Isoflavones regulate cAMP response element-mediatedtranscription1). Our last work shows that estrogen has effect through ERE-independent pathway,such as estrogen can inhibit CRE gene transcriptional activity. So we transfected CREreport gene into MG63cell line, then treated cells with different dose of estrogen, daidzeinand genistein. It was found that estrogen inhibit CRE report gene transcriptional activity indose dependent manner, daidzein and genistein （10-6M）also have inhibition effects, butcan’t stimulate CREB phosphorylation. When treated with exogenous CRE agonist,8-Br-cAMP, it was found that CRE report gene transcriptional activity increase; butestrogen and genistein can block the effect of8-Br-cAMP, daidzein can’t do that.2). We further investigated effects of daidzein and genistein on SGK-1and IL-8mRNA expression in MG-63cells, because the promoter of these two genes have CRE-likesequence. It was found that both estrogen and genistein can inhibit these two genes mRNAexpression, but daidzein can only inhibit IL-8mRNA expression. Firstly induced with8-Br-cAMP to increase level of SGK-1and IL-8mRNA expression, and then treated withE2, G and D, the results were same as no use of8-Br-cAMP. All above suggests that D andG have estrogen-like effects, that is D and G can change gene transcription throughregulating CRE-like sequence of target gene promoter. The regulating mechanism of D andG may be different in different target genes.3). Then, MG63was co-transfected with ER or ER and CRE report gene, andtreated with E2, D, and G. We found that all three can inhibit CRE transcriptional activity indose dependent manner when ER and CRE co-transfected. On this basis, we usedICI182,780,4-OH-tamoxifen and ER specific antagonist MPP, and found that ICI andMPP stimulate CRE gene transcriptional activity through ER by themselves,4-OH-tamoxifen has no effects. But when they respectively act with E2or D and G, all canreverse inhibition effects of E2, D and G on CRE report gene transcriptional activity. When cells were co-transfected with ER and CRE report gene, E2has no effects onCRE report gene. However, D and G still inhibit report gene transcriptional activity in doseindependent. ICI182,780,4-OH-tamoxifen and ER specific antagonist THC can’tinfluence transcriptional activity of CRE report gene. But co-treated with D and G, they allcan reverse inhibition effects of D and G on CRE report gene transcriptional activity.Conclusion:1. RSG can significantly inhibit differentiation of calvarial osteoblasts. SHP-1canstimulate differentiation. Both of them have effects through GSK3pathway. RSGstimulates GSK3activity to increase degradation of β-catenin; SHP-1stimulatesproliferation through down-regulating GSK3activity. And RSG inhibits SHP-1mRNAexpression in calvarial osteoblasts. The inhibition of RSG is partly through PPAR.Meanwhile, GPR40remains low lever during IM, RSG can stimulate GPR40proteinexpression which have parts of RSG effects.2. RSG also can inhibit proliferation of calvarial osteoblasts. SHP-1has positiveregulation to proliferation. Those effects are also through GSK3pathway. And RSGinhibits proliferation through PPAR-independent pathway. It plays role through GPR40.3. Effects of different type of phytoestrogen are not similar when binding with ERs. Dand G have estrogenic activity in MG-63cells, which can stimulate transcriptional activityof ERE report gene and inhibit transcriptional activity of CRE report gene as estrogen dose.And puerarin has anti-estrogenic activity in MG-63cells.4. There are different transcriptional regulations to CRE report gene through differentsubunits of ER. Estrogen regulates CRE gene transcriptional activity mainly through ER.D and G regulate CRE report gene through both ER and ER.