Osteogenic And Adipogenic Differentiation Mechanisams Of Human Mesenchymal Stem Cells Induced By 17β-estrogen

Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture.More and more countries face the problem of aging society for the rapid population aging which is mainly caused by migration, longer life expectancy (decreased death rate), and decreased birth rate, leading to that the osteoporosis becomes a major clinical problem for golden citizens and the society. The osteoporosis has a multifactorial etiology and a complicated pathogenesis and estrogen deficiency is considered to be the leading cause of and play a pivotal role in osteoporosis.Estrogen is a very important member of steroid hormone which also includes progestogen and androgen. The osseous tissue is a major target by estrogen and clinical and laboratory evidence shows that the estrogen promotes cartilage morphogenesis, bone maturation, the fusion of osteoepiphysis growth plate and osteoblast differentiation during bone remodeling. So the investigation of the role of estrogen in osteoporosis is very necessary.Recent works find that the osteoporosis shows both osteopenia and lipotrophy inside the bone marrow. For patients who are suffering osteoporosis, the differentiation pathway of the hMSCs (human mesenchymal stem cells) could be altered, with the decreased potential of osteogenic differentiation and increased potential of lipogenic differentiation,hMSCs are multipotential stem cells derived from mesoderm, possessing potent proliferative capacity and self-renewal and multilineage differentiation. hMSCs can differentiate into osteoblasts,chondrocytes,adipocytes,myocytes,hepatocytes and stroma cells under proper in vitro and in vivo environments.To elucidate whether the different estrogen concentrations cause the alteration in differentiation pathway and capacity, a series of experiments designed to investigate the possibly regulated signal transduction pathway by estrogen in the osteogenic differentiation and adipogenic differentiation of hMSCs, aiming to provide a new perspective for treatment. Morphology and molecular biology approaches are applied to detect the influence of estrogen on morphology and function of hMSCs and possible mechanisms. Also, the involvement of PI3K/AKT/Cofilin signal transduction pathway in the osteogenic and adipogenic differentiation of hMSCs is measured by RT-PCR,Western blot and immunostaining methods.Results:1. hMSCs of high purity were isolated and obtained by the density gradient separation method and adherent culture system. A standardized platform has been established for isolation, cultivation, purification, identification and amplification of hMSCs. The hMSCs under in vitro culture maintain stable biological characteristics at even passage 12 and stay at the state of undifferentiation, providing an excellent cell source for extended experiments 2. After placed into osteogenic medium, hMSCs exhibit significant changes in cell structure, modulating from elongated spindle shape which is the characteristic appearance of the hMSCs to a polygonal shape. Immunostaining result shows that a proportion of hMSCs population positively expresses alkaline phosphatase (ALP) which is a specific protein marker of osteoblast and almost all of the hMSCs population positively expresses ALP at day 7. The hMSCs gather together like nodosity at day 14, and positive expression of ALP by immunostaining and deposition of calcium salts by alizarin reds staining are observed. Successful induction of osteogenic differentiation is indicated by the formation of characteristic deposition of calcium salts at day 22 to 24. In contrast, the osteal nodule is not observed with a positive expression of ALP by immunostaining and deposition of calcified matrix by alizarin reds staining when the ICI182, 780(it is a steroidal pure anti-oestrogen devoid of agonist activity) and LY294009 are added to osteogenic medium respectively. In control group, proportional population of hMSCs changes the appearance from elongated spindle-shape to a polygonal shape, with a positive expression of ALP by immunostaining and no deposition of calcified matrix by alizarin reds staining.3. RT-PCR analysis of mRNA expression for osteocalcin (OCN) shows that the expression of OCN mRNA increases strikingly with treatment of estrogen and the stimulatory effect on transcription resulting from treatment of estrogen could be attenuated by the anti-estrogen ICI182,780 and LY294009 , resulting in a significant decrease in the OCN mRNA expression. The OCN mRNA expression can't be detected in the control group. On the basis of these experiments we conclude that estrogen is very important in the osteogenic differentiation of hMSCs4. Immunofluorescence and confocal laser scanning microscope images show that the microfilament becomes short and are cross-linked to each other after treatment of estrogen to induce osteogenic differentiation of hMSCs. The appearance of the microfilament of estrogen induction group is distinct to that of control group. Both ICI182,780 and LY294009 inhibit the structural changes of microfilament in estrogen treated group. On the basis of these experiments we conclude that estrogen plays its role in osteogenic differentiation of hMSCs through changing the arrangement of microfilament5. Oil red O staining shows that estrogen strikingly inhibits the lipogenic differentiation of hMSCs, and its inhibitory effect on lipogenic differentiation is counterstriked by the anti-estrogen ICI182,780.6. After lipogenic induction for 12 days, RT-PCR analysis of mRNA expression for PPARγ-2 shows that estrogen exerts a significantly inhibitory effect on mRNA expression of PPARγ-2 which is specific to lipocyte, and that the inhibitory effect can be antagonized by ICI182,780, resulting in a striking increase in the mRNA expression of PPARγ-2. On the basis of these experiments we conclude that estrogen inhibits the lipogenic differention of hMSCs.7. After lipogenic differentiation, the morphology of microfilament changes significantly, depolymerizing from F-actin to G-actin, and also the lipid droplet is observed in cytoplasm. It suggests that estrogen might inhibit lipogenic differentiation through the depolymerization of microfilament.8. Estrogen at concentrations of 10-7M and 10-6M strikingly promote the proliferation of hMSCs. 9. Flow cytometry analysis shows that estrogen strikingly inhibits apoptosis of hMSCs. Both ICI182, 780 and LY294009 significantly promote the apoptosis.10. Western blot analysis shows that the expression of P-cofilin is significantly lower than that of control group when hMSCs treated with estrogen for 30 minutes, and it can be antagonized by both ICI182,780 and LY294009. We then measures the expression pattern of P-AKT and find that estrogen significantly promotes the expression of P-AKT, and also that this stimulatory effect can be inhibited by both ICI182,780 and LY294009. On the basis of these experiments we conclude that estrogen promotes the proliferation and inhibit apoptosis of hMSC by regulating the expression level of P-cofilin trough PI3K/AKT signal transduction pathway, thus playing its important role during the proliferation stage of osteogenic differentiation of hMSCs.11. We construct the siRNA vector plasmid pSUPER-SSH1L and transfect it into hMSCs and then treat hMSCs bearing plasmid pSUPER-SSH1L with estrogen. It shows that, after silencing of SSH1L gene, the proliferation rate of hMSCs treated with estrogen is lower than that of control group which doesn't contain siRNA vector plasmid pSUPER-SSH1L. It confirms that estrogen promotes the proliferation of hMSC by regulating the expression level of P-cofilin trough PI3K/AKT signal transduction pathway.12. We analyze the expression level of P-cofilin by western blot at the maturation stage of osteogenic differentiation of hMSCs and find that, compared to control group, estrogen strikingly promotes the expression level of P-cofilin. On the basis of these experiments we conclude that estrogen modulate the change of the microfilament through regulating the expression level of P-cofilin, thus promoting the bone maturation.13. During lipogenic differentiation, the estrogen promotes the expression of P-cofilin and inhibits the depolymerization of F-actin to G-actin, thus inhibiting the lipogenic differentiation of hMSCs.Collectively, all of our detailed works elucidate that estrogen promotes the osteogenic differentiation of hMSCs and inhibits the lipogenic differentiation of hMSCs, and the possible mechanisms. Osteogenic and lipogenic differentiation of hMSCs are the most two important perspectives in osteoporosis. So our works elucidate the role of estrogen in osteoporosis and provide the laboratory basis for the development of new therapeutic strategies targeting the signal transduction molecules during osteogenic and lipogenic differentiation of hMSCs.