CKIP-1 Negatively Regulates The Osteogenesis Differentiation Of Mesenchymal Stem Cells

Objective:Osteoporosis is a typical disease of metabolic bone diseases, and the basis of the disease is that the dynamic balance of bone formation and bone resorption is broken. CKIP-1 is a protein that is recently paid lots of attention to, and it is involved in many physiological activities. One of its important functions is negatively regulating the process of osteogenic function, through enhancing the ubiquitin ligase E3 activity o f Smurf1. Bone marrow mesenchymal stem cells are one kind of multiple differentiation potential of cells, and they are also the precursor cells of osteoblasts, regulating whose function can affect the osteogenetic differentiation, which affects the body’s bone metabolism. Our project is to use the model of gene knockout mice, to detect how CKIP-1 affect the mice’s bone on general level, how CKIP-1 affect the cells’ biological characteristics on cell level, and how CKIP-1 acts its negatively regulation in osteogenetic process on the molecular level, respectively. In order to find that how a protein can negatively regulate the bone formation, to further open out the possible mechanism of bone metabolization, which provides experimental evidances for the research of metabolic bone disease such as osteoporosis.Methods:1. By establishing CKIP-1 gene knockout mice breeding system and using thepolymerase chain reaction(PCR) to identify gene type of the new-born mice. I measured the increase of the weight and length of their tails, in order that I can evaluate the general level of their development condition.2. I mensurated the thighbone, vertebrae and mandible parameter of WT(CKIP-1+/+) and KO(CKIP-1-/-) mice by Micro-CT scan, and also I observed the morpho logy strcture and relevant protein expression by histological staining method.3. I cultivated the bone marrow mesenchymal stem cells of WT and KO mice in vitro, detected the expression of surface marker molecule through flow cytometry, and observed the difference between the multi-directional differentiation ability to ensure that the cells cultivated were stem cells coming from the mesenchymal tissue.4. I used MTT and flat cloning experiments methods to detect the ability of proliferation of WT and KO mice’s BMSCs, flow cytometry to detect the cell cycle and apoptosis, to test whether CKIP-1 deficiency had effects on biological characteristics of stem cells.5. I used osteogenesis and adipogenic liguid to make the cells differentiate to bone and fat in order that I coule detect their difference of the ability between WT and KO mice’s BMSCs. The alkaline phosphatase activity had also been detected. And I used the nude mice to set up a model of skull defeci, through which I could evaluate the different ability of osteogenesis in vivo by mixing the cells and fibrin glue together. After transplant about 10 weeks, HE staining, Masson trichromatic dyeing and Micro-CT scan were used to evaluate the condition of the new bone formation.6. After induction toward osteogenesis, I used the application of real- time quantitative polymerase chain reaction(PCR) and western blot method to detect osteogenesis related molecular changes in the m RN A and protein expression level, to find out through which protein CKIP-1 affected the process of bone formaion.7. Using Western Blot method to detect related proteins of MAPK pathways in WT and KO mice BMSCs after osteogenesis induction, explore regulatory changes in the process of osteogenesis CKIP-1 channel proteins and its phosphorylation to find a possible mechanisms of this phenomenon.Results:1. The mouse tail genotype identification of coarse formulation combined with PCR amplification, the KO mice didn’t express exon 3 of CKIP-1 and it expressed artificial add neo resistance genes. I choosed the nest single-sex WT(CKIP-1 +/+) and KO(CKIP-1-/-) mice for follow- up experiments, the quality of good appearance for subsequent reproduction. Furthermore, since the WT and KO mice after birth to adult in the process, its tail long, weight, all in accordance with the linear rule, two groups did not reflect the difference(P > 0.05).2. According to the results of Micro-CT scan, the BV/TV and Tb.N of thighbone and vertebra were more in KO mice than in WT mice(P < 0.05), and BS/BV and Tb.Sp of thighbone and vertebra were less in KO mice than in WT mice(P < 0.05). However, no difference was observed in mandible(P > 0.05). The HE staining results showed that there was no significant difference in their bone general tissue structure between WT and KO mice. The immunofluorescence staining showed CKIP-1 expressed weakly in thighbone, vertebra and mandible of WT mice, also CKIP-1 expressed in dental tissue, expecially in dentine. No staining of CKIP-1 was found in KO mice.3. I cultivated the BMSCs in thighbone of WT and KO mice after collagenase digest, the experimental model was successfully established. And the flow cytometry results showed the cells cultured expressed CD90, C D105 and sca-1, which were expressed in cells coming from mesenchymal tissue, and they didn’t express CD31 or CD45, which were expressed in cells coming from hematopoietic system. Those CDs expressed showed no differences between WT and KO cells(P > 0.05). After osteogenesis and adipogenic induction, the KO mice’s BMSCs had a much higher ability in osteogenesis and adipogenic process(P < 0.05).4) Results of MTT and tablet cloning experiments showed the KO group had a faster speed in proliferation than WT ones(P < 0.05), and the ability of cloing was also stronger(P < 0.05). The results of flow cytometry revealed that the S period cells of KO group were more than WT ones, which showed they had a stronger ability of proliferation(P < 0.05). However, no evidence wa s found of their apopotosis ability between WT and KO groups(P > 0.05).5. After osteogenesis induced, BMSCs in KO mice showed stronger osteogenesis ability in vitro, and its alizarin red staining and ALP staining results were significantly higher than that of WT group(P < 0.05); And the alkaline phosphatase activity of KO group of cells was much higher than WT group(P < 0.05). In N ude mouse skull defect experiment model, KO group of cells in vivo osteogenesis ability was stronger than the WT group of cells, formation of osteoid more(P < 0.05) and the structure is more mature.6. In transcription level, after osteogenesis induction, CKIP-1 expression dropped, and transcription factors osterix and Runx2, ALP, Col, BSP and OCN expression were significantly enhanced, KO group is higher than the WT group(P < 0.05). Protein level was in line with the experiment, the cells of KO group had a higher level expression of bone related factors than the WT group(P < 0.05).7. Related proteins expression and its phosphorylation condition of MAPK pathway had been detected, and results showed that WT mice’s BMSCs expressed CKIP-1 protein, and KO groups didn’t. After osteogenesis induction, KO group expressed a lower level Smurf1 than WT group(P < 0.05), while MEKK2 expression was significantly enhanced(P < 0.05), JNK, p-JNK, p-c-jun, p38 and p-p38 expression were also increased(P < 0.05). Nevertheless, ERK1/2, p- ERK1/2 were not detected during this period.Conclusions:1. It is a relatively precise, simple and effc ient method to identify the genotype of mice by the mouse tail roughly extration combined with PCR. The absence of CKIP-1 didn’t affect the birth and development of mice, and the reproduction in mice followed by Mendel’s law, which can be a stable genetic breeding system.2. The KO mice seemed stronger in general bone quality, and a more optimized structure than in WT ones. However, there was no obvious difference in mandible. CKIP-1 was also expressed in dental tissue, pointing out that it may be associated with teeth development.3. CKIP-1 didn’t influence the expression of stem cell surface markers and molecular and stem cell apoptosis; and could inhibit the stem cell cloning formation ability and proliferation rate.4. CKIP-1 plays its role by inhibiting the expression of osteogenesis related molecules in m RNA and protein expression level, which can negatively regulate the osteogenesis of stem cells. The process is due to activating the MEKK2, then causing JNK, c-jun and p38 phosphorylation and implementation.In conclusion, CKIP-1 is an important protein in the process of osteogenesis by negatively regulating bone formation. The influence of stem cells’ function and related mechanism, may provide a new method for the bone metabolism related disease in the future.