Regulatory Effect Of IGFs Family On Osteogenesis

There were many growth factors taking part in the control and regulation of metabolism within the bone development. 2,3,7,8 - tetrachlorodibenzo - p -dioxin (TCDD) is a widely spread environmental contaminant with extremely toxic effect. TCDD influence the activity of diverse hormones, hormone receptors, mitogens, and other biological response modifiers and these interactions undoubtedly play an important role in the toxicity of these response. Retinoic acid (RA) is ramification of Vitamin A, having extensive biological effect on controlling conformation genesis, proliferation, differentiation, growth, metabolism of kinds of tissues and cells and stabilization of internal environment. The laboratory animal studies on TCDD and RA exposure indicated high incidences of congenital developmental malformations. In our previously study, we analyzed gene expression profile in IGF gene family of TCDD - and RA - reduced fetal rats as well as cells and found antiestrogenic effect of TCDD and RA on IGF genes. In the present study we suggested the evidences of TCDD - and RA - induced toxicity to the osteogenesis of rats, whereas examined the antiestrogenic effect of TCDD through altering transcription of IGFBP - 6 gene in vitro and in vivo. These results might provide the new basis of theory and pathway of investigation to interpret the teratogenesis of TCDD, RA and its mechanism of action.OBJECTIVE: To investigate the toxic effect of environmental teratogen and RA on the skeletal development; To investigate the antiestrogenic effect of enviroment teratogen on the gene expression of IGFs family in osteoblast cells; To interpret the mechanism of TCDD acted as antiestrogenic effect through altering IGFBP - 6 gene expression.METHODS: The Wistar fetal rats with congenital skeletal malformation were induced by TCDD (10-15 ug/kg) and RA (100 - 140 mg/kg). On pregnant day 10, rats were injected with TCDD and (or) RA dissolved in the mineral oil by gastric canal. On pregnant day 20, fetal rats were taken out, andtheir developments were observed and recorded. The MC - 3T3 - El cells were treated with lOnM TCDD, IuM RA and /or 1uM estrogen (17 - B - estradiol, E2) , when the cells grew to the logarithm growth phase for 24 hrs. The cell proliferation was observed with flow cytometer and MTT colorimetric test. The insulin - like growth factor - 2 (IGF - 2 ) and IGF conjugated protein - 6 (IGFBP -6) mRNA level in rat calvaria bone tissue and MC -3T3 - El cells were detected by northern blotting analyses and reverse transcription polymerase chain reaction. The estrogen responsive element ( ERE) in IGFBP - 6 gene promoter was identified and involved in TCDD - reduced regulation of the gene expression by electromobility shift assays (EMSA). The ERE oligonucleotides were chemically synthesized, annealed, and labeled at the 5' - ends by polynucleotide kinase and r - 32P ATP (3000 Ci/mmol). For competed experiment the 400 fold molar excess of the cold ERE oligonucleotides were added to reaction mixture. The protein nucleic acid complexes were identified by autoradiography for 48 h. The pSG5HEO and IGFBP -6 promoter pCAT vectors were transfected into Cos -7 cells with Iipofectamin2000; These Cos - 7 cells where treated with 1 jxM E2, with lOnM TCDD and with both lOnM TCDD and 1 jxM E2, individually;The Cat protein was examined by CAT - ELS A.RESULTS: The congenital skeletal defects in fetal rats were induced with 10 - 15ug/kg TCDD or with 100 - 140 mg/kg RA with dosage - dependence effect. The IGFBP - 6 mRNA in rat calvaria tissue and MC - 3T3 - El cells were increased with TCDD and (or) RA, whereas the cell proliferation and IGF -2 mRNA in MC -3T3 - El cells was decreased in the status of E2. The ratio of ( S + G2)/( G2 + M + S) and PI value of MC -3T3 - El cells increased in the condition of 1um/L E2. The effects of E2 on IGFBP -6 and IGF -2 gene expressions were suppressed by TCDD and RA. The binding of ERE at hIGFBP -6 5' -UTR (DNA sequence: AACTCTGACC, +94 to +103) was enhanced by TCDD. Results of this experiment showed reta...