The Role And Mechanism Of Gap Junctional Intercellular Communication In Osteoblasts Exposed To17-β Estra Diol

Backgrounds:Estrogen has garnered considerable attention because of its importance in bone mass maintenance and the efficacy of hormone therapy in combating postmenopausal osteoporosis, and it has been demonstrated to induce osteoblast proliferation and bone formation. Gap junctions are membrane spanning protein channels that are composed of two juxtaposed hemichannels present on the surfaces of adjacent cells. Gap junctions enable neighboring cells to physically link and facilitate intercellular communication by allowing passage of small molecules from cell to cell quickly in a process known as gap junctional intercellular communication, which makes the relevant cells function as a whole. In the previous experiment of our project, appropriate concentration17-β estradiol applied on MC3T3-E1cells and the results of microarray showed that Gjal in the gap junctions pathway was elevated significantly, demonstrating that gap junction pathway plays important roles in the response of osteoblasts to17-β estradiol. This project may provide theoretical foundation for improvement in wound healing quality, the molding and remolding of alveolar bone and treatment of bone diseases.Objective:On the above foundation, this study is to explore the role and mechanism of gap junctional signal pathway in the estradiol-induced biological response in MC3T3-E1cells, in order to make clear the molecular mechanism of the biological reaction of osteoblasts under17-β estradiol further. This project may provide theoretical foundation for tissue engineering, development in wound healing quality, treatment of bone diseases and exogenous gene therapy.Methods:1. MC3T3-E1cells were cultured in vitro.2. Cells were divided into four groups randomly, which were control group,17-β estradiol group (10-8mol/L), inhibitor group (30μ mol/LAGA) and17-β estradiol combined with inhibitor group.3. Methyl thiazol tetrazolium (MTT) and alkaline phosphatase (ALP) were adopted to test the proliferation and differentiation of osteoblasts.4. Alizarin red staining and calcium ion concentration were used to test the differentiation and osteogenesis ability of osteoblasts.5. RT-PCR and Western blot were conducted to detect the protein and mRNA expression level of Runx2after corresponding treatment.6. Statistically analyses were conducted through SPSS17.0software.Results:1. MC3T3-E1cells were in good conditions.2.17-β estradiol increased the MTT activity compared with control groups (P<0.05); the MTT activity decreased with the application of AGA both in17-β estradiol and static groups (P<0.05).3.17-β estradiol increased the ALP activity compared with control groups in3d (P<0.05), while ALP activity were not significantly changed in5d (P>0.05); the ALP activity decreased with the application of AGA both in17-β estradiol and static groups (P<0.05); the ALP activity increased obviously in5d groups compared with the corresponding3d groups.4. The number of calcified nodules with Alizarin red staining:17-β estradiol group>control group>inhibitor group>17-β estradiol combined with inhibitor group.Calcium ion concentration:17-β estradiol group>control group, inhibitor group>17-β estradiol combined with inhibitor group (P<0.05); increased in17-β estradiol groups compared with control groups (P<0.05); calcium ion concentration decreased with the application of AGA both in17-β estradiol and static groups (P<0.05). 5. The expression level of Runx2mRNA increased1.2times in17-β estradiol groups compared with control groups (P<0.05); the expression level of Runx2m RNA decreased with the application of AGA both in17-β estradiol and static groups (P<0.05); the expression level of Runx2mRNA increased2times in the17-β estradiol+AGA groups compared with control groups (P<0.05).6.17-β estradiol increased the protein expression level of Runx2compared with the control group(P<0.05); treatment of gap junctional signal inhibitor leaded to the marked decline the protein and mRNA expression level of Runx2of both in17-β estradiol and static group(P<0.05); the expression level of Runx2protein increased slightly in the17-β estradiol+AGA groups compared with control groups, which is statistically significant (P<0.05).Conclusions:1. Appropriate concentration and duration of17-β estradiol can regulate the proliferation and differention of MC3T3-E1cells through Cx43-mediated gap junctional intercellular communication.2. Gap junctional intercellular communication is the signaling pathway that can regulate the activity of osteoblasts, but not the only one. The specific regulatory mechanism has yet to be researched.