Inflammation-Mediated Age-Dependent CKIP-1 Effects On Osteogenesis In Mesenchymal Stem Cells

Objective: Osteoporosis is a silent disease which can significantly increase an individual’s risk of fracture. Fractures can be physically debilitating and consequently cause a financial burden on the patient, so it is key to treat osteoporosis before a fracture occurs. Bone Marrow Mesenchymal Stem Cells(MSCs) are mesoderm derived adult stem cells defined by their self-renew ability and multipotent differentiation ability, which play a key role in regulation of balance between bone absorption and formation, The PH domain-containing protein casein kinase 2 interactingprotein-1(CKIP-1, also known as PLEKHO1) was originally identified as an interacting protein of CK2 kinase. CKIP-1 was a negative regulator of osteogenesis. CKIP-1 interacts with Smurf1 resulting in an increase in its E3 ligase activity. However, the effect of CKIP-1 on osteoblast precursor mesenchymal stem cell(MSC) remained unknownMethods: 1. To established CKIP-1 gene knockout mice breeding system. using Polymerase chain reaction, PCR-agarose gel electrophoresis to identified the offsping mice.Determina- tion of CKIP-1 gene knockout mice and wild-type mice in the breeding ability and body weight, analysis of gene knockout mice development. in order to evaluate the general level of their development condition. 2.We cultivated the bone marrow mesenchymal stem cells of WT(CKIP-1+/+) and KO(CKIP-1-/-)mice in vitro, cell phenotype was analyzed by flow cytometry. MTT viability assay, clone-forming ability was used to study the proliferative ability. Osteogenic and adipogenic induction were performed. The osteogenic ability was find by Alizarin red staining, ALP staining and ALP activity. Quantitative Real-time PCR determined the m RNA expression levels of osteoblast marker genes, including Runx2 and OCN. The adipogenic ability was detected by Oil red O staining. 3. Micro-computed tomography(CT) imaging analysis was performed in WT and KO mice between 2 months and 18 months. To observe the differences of bone imaging parameters(BV/TV,BSA/BV,TB.N,TB.Sp).we analysis the difference in content of the bone. 4. We used real-time quantitative PCR and Western blot respectively to detect the localization of CKIP-1 protein for 2 months and 18 months of C57BL/6J mice in vivo and vitro. Whether there are the different in CKIP-1 during in the process of aging. 5. Wild type mice were injected with IL-1β respectively for 2 months and 18 months. We used real time PCR and Western blot to detect the difference in CKIP-1 expression. We further investigate the relationship between CKIP-1 and inflammation.Results: 1. To successfully established CKIP-1 gene deficient mice, we used PCR amplification to identify the gene. Knock out CKIP-1 gene mice didn’t express exon 3 of CKIP-1. Age and gender matched, the same nest single-sex WT and KO mice were chosen for follow up experiments. The WT and KO mice after birth to adult, reproductive ability, length of their tails, in accordance with the linear rule.(P>0.05)As the mice aged, the difference in body weight between the groups gradually increased. At 12 months, the average weight of the WT mice was 33 g, but the average weight of the KO mice was 39 g(P<0.05). At 18 months, the weight difference increased to 11 g(P<0.05). 2. We isolated MSCs in vitro from CKIP-1 KO and WT mice. The CKIP-1 KO MSCs retained typical fibroblastic spindle shapes and maintained the ability to form CFUfibroblasts. We used flow cytometry to characterize the surface molecules of CKIP-1 KO and WT MSCs. Both MSCs subsets showed characteristic mesenchymal surface markers, including CD90, CD44, and CD106. After culturing in osteogenic differentiation medium for 21 days, both MSCs subsets formed distinct nodules as shown by Alizarin red staining in an induced time-dependent manner. The CKIP-1 KO MSCs produced a higher amount of extensive calcified deposits than the WT MSCs. ALP activity was detected after osteogenic induction for 7 days. The ALP activity in CKIP-1 KO MSCs was higher than that in WT MSCs(P<0.05). The quantifications of the amount of Alizarin red staining and ALP activity showed that the osteogenic capacity of CKIP-1 KO MSCs was significantly stronger than that of the WT MSCs(P<0.05). The quantitative PCR analyses showed that the expressions of the osteogenic marker Runx2, which is a critical transcription factor for osteoblastic differentiation, and osteocalcin(OCN), which is a mature osteoblast marker were significantly enhanced,CKIP-1 KO MSCs is higher than CKIP-1 WT Mscs(P<0.05). Under adipogenic induction conditions for 6 days, both subsets of MSCs were able to form Oil red-O-positive lipid droplets. However, the amount of Oil red O-positive lipid droplets in CKIP-1 KO MSCs was significantly higher than in WT MSCs. Adipogenic differentiation was further confirmed by the increased expression of specific adipogenic markers, including PPARγ, as determined by RT-PCR. We observed no significant differences between the MSCs. Adipogenesis was also enhanced in CKIP-1 KO MSCs compared with WT MSCs(P<0.05). 3. Micro-CT analyses of trabecular bone from 2-month-old CKIP-1 KO mice showed no significant changes in BV/TV 、BSA/BV、TB.N、TB.Sp compared with the WT mice(P>0.05). However, the micro-CT analysis of the 18-month-old CKIP-1 KO mice showed higher BV/TV 、 and Tb.N compared with the WT mice(P<0.05).18-month-old CKIP-1 KO mice showed lower BSA/BV、TB.Sp compared with the WT mice(P<0.05).4. We evaluated the expression of CKIP-1 in WT mice. Quantitative PCR results showed that the CKIP-1 gene expression from the MSCs derived from 2-month-old mice was not significantly different from that of the MSCs derived from 18-month-old mice(P>0.05). Under osteogenic induction conditions, CKIP-1 expression was also not significantly different between the different age groups(P>0.05). Western blot analyses showed that the expression of CKIP-1 could be detected but was weak in both MSCs derived from young and old mice in normal or osteogenic induction. We acquired bone marrow in vivo from 2- and 18-month-old mice to further detect the expression of CKIP-1. Quantitative PCR detected 4.3-fold higher gene expression levels in 18-month-old mice compared with 2-month-old mice(P<0.05). Western blot results also showed that the CKIP-1 expression in old mice was significantly higher in young mice(P<0.05). 5. Quantitative PCR results showed that in vitro IL-1β significantly up-regulated the expression of the CKIP-1 gene in MSCs derived from young and old mice(P<0.05). Western blot analyses showed that the expression of the CKIP-1 protein in inflammation-induced MSCs was higher compared with normal MSCs(P<0.05).To further detect the effect of inflammation on CKIP-1 expression, we regularly injected IL-1β into 2-month-old mice to simulate chronic inflammation. At 3 months, we assayed the CKIP-1 expression in the bone marrow of mice. The quantitative PCR results showed that CKIP-1 expression in the IL-1β injection group was 3.2-fold higher than that in the sham-injected group(P < 0.05). Western blot analyses also showed that the IL-1β up-regulation stimulated CKIP-1 up-regulation in vivo(P<0.05). In addition to CKIP-1 expression, adult sham-injected mice were slightly larger in body size and heavier in body weight. Micro-CT analyses of the IL-1β injection group showed decreased femur bone masses compared with the sham-injected mice(P<0.05).Conclusions: CKIP-1may play an important role in regulation of the adipogenic and osteogenic differentiation of bone marrow mesenchymal stem cells(MSCs). The aging might regulate CKIP-1 expression to play a role in regulation of osteogenic differentiation. These results indicate that inflammation in MSCs is critical in decreasing bone mass through the up-regulation of CKIP-1 expression.