Study On The Mechanism Of Dehydroepiandrosterone (DHEA) In Metabolism Of Vitamin D

Part1 Isolation and Identification of Human Marrow Stromal CellsAccording to the established protocols in Julie’s lab, I kept using this culturing system for collecting and culturing human bone marrow stromal cells (hMSCs), and it provides plenty of cells from different patients for the following experiments. Human bone marrow samples were obtained with IRB approval from femoral tissue discarded during hip replacement surgery in Brigham and Women’s hospital. Low-density mononuclear cells were isolated by density centrifugation on Ficoll/Histopaque 1077. Adherent hMSCs were isolated and identified. Human MSCs were cultured and passaged ina-MEM (10% FBS-HI). The cells can be induced into different cell types with osteogenic, adipogenic, or chondrocyogenic medium separately in different time points. ALP activity and Alizarin Red S staining were conducted for identification of osteogenesis; Oil Red-O staining were used for identification of adipocytogenesis; and Alcian blue staining was for identification of chondrocytogenesis. The primary hMSCs will attach to the dishes after 6-24 hours after culturing, the shape looks like spindle or shuttle. The clones of hMSCs formed after 1-3 days. The cells proliferated rapidly after 1 week culture. When the cells were almost confluence, the appearance of the cells was like fibroblasts. The cells could be passaged for 4-12 times, each passage might need 1-2 weeks. ALP activity and Alizarin Red S positive area could be detected after the cells were cultured in osteogenic medium. Human MSCs could differentiate to Oil Red-O positive cells after adipocytogenic culture. Human MSCs after chondrocytogenic induction that were positive for Alcian blue staining were chondrocytes.In sum, hMSCs can be isolated from bone marrow, and can be expanded, cultured and passaged stablely. Human MSCs can differentiate into osteoblasts, adipocytes, or chondrocytes in different culture medium.Part 2 DHEA regulates IGF-I signaling and osteoblast differentiation in human mesenchymal stem cellsHuman bone marrow-derived skeletal stem cells a.k.a mesenchymal stem cells (hMSCs)have been shown to be precursors of several different cellular lineages, including osteoblast,chondrocyte, myoblast, adipocyte, and fibroblast. Several studies have shown that IGF-I could stimulate osteogenic differentiation of mesenchymal stem cells (MSCs), thus maintaining proper bone microarchitecture and mass. Moreover,experimental evidence from animal models and studies on human has shown that DHEA supplementation significant increases in serum IGF-I and DHEA was shown to stimulus IGF-I gene expression directly in rat granulosa cells.In this study, We tested the effects of treatment with DHEA on IGF-I gene expression in hMSCs. The results show that the peaking increases in IGF-I gene expression at 4-6 hours and the optimum concentration range is lOnM. And then, we tested mechanisms by which DHEA activation of IGF-I signaling pathway and whether these pathways activate during osteoblast differentiation of hMSCs. With selective small chemical kinase inhibitors, we demonstrated that IGF-I requires PI3K,P38 MAPK,P4t2/44 MAPK to active osteoblastogenesis in hMSCs. Block of PI3K,P38 MAPK,P42/44 MAPK pathway with small chemical kinase inhibitors inhibited alkaline phosphatase activity and antagonized the active effects of DHEA on ALP,BSP,RUNX2,COL 1 expression, suggested that DHEA cooperated with IGF-I signaling in active of osteoblastogenesis in hMSCs. In summary, we found that there was a time and dose-dependent effect of DHEA on expression of IGF-I in hMSCs.DHEA activates IGF-1 signaling pathway via P38 MAPK,P42/44 MAPK and PI3K pathways, and modulates osteoblastogenesis via P38 MAPK,P42/44 MAPK and PI3K pathways in hMSCs;Part 3 Effects of DHEA on Human Marrow Stromal Cells: Role of Vitamin D Metabolism25OHD3 can be transformed into 1,25(OH)2D3 by la-hydroxylase/ CYP27B1 in human bone marrow mesenchymal stem cells. osteogenic differentiation capability of human bone marrow-derived mesenchymal stem cells can be promoted through the enhancement of 1,25 (OH) 2D3 synthesis by 1a-hydroxylase/CYP27B1. This study was designed to define the effects of age on la-hydroxylase/CYP27B1 in hMSCs and other related gene expression. A series of hMSCs obtained from 19 subjects (40 to 87 years, mean age 56±11 years) was used to determine expression of CYP27B1/1a-hydroxylase. The mean level of expression of CYP27B1 in the older group (>55 years, n=12, p=0.007) was 64.4% of that for the younger group (<50 years, n=7). There was an inverse correlation between CYP27B1 expression and age (r=-0.478; p=0.007). However, the expression of osteogenic marker are upregulated slightly by 25OHD3 t in the elderly group, but 25OHD3 and 1,25 (OH) 2D3 can promote Differentiation of human marrow stromal cells (hMSCs) to osteoblasts after pretreatment of DHEA In the older group,We used specific inhibitors to identify whether cAMP response element binding protein (CREB) and insulin-like growth factor I (IGF-I) mediate DHEA effects in hMSCs. DHEA provided hMSCs with responsiveness to 25OHD3 and with phases of 1,25(OH)2D3 synthesis, of CYP27B1 upregulation, and of CREB activation, and increases by DHEA in CYP27B1 expression were obliterated by KG-501, which specifically inhibits the downstream binding of activated CREB. the peak of CREB signaling was diminished by AG1024, an inhibitor of IGF-I receptor. Thus, CYP27B1/1a-hydroxylase increase is mediated by IGF-I and its activation of CREB.In sum, DHEA provided hMSCs with responsiveness to 25OHD3 by upregulating expression of CYP27B1 and did so through CREB and IGF-I pathways.