ROCK-LIMK-Cofilin Axis Regulates Osteoblast Proliferation,Migration And Differentiation

Purpose: To investigate whether ROCK-LIMK-Cofilin axis mediated cytoskeleton changes play a regulatory role in osteoblast proliferation,migration and differentiation.Methods: 3T3-E1 were treated with ROCK agonist and LIMK inhibitor.Firstly,the appropriate concentration and time of treatment were selected.Then the cells were divided into three groups: ROCK agonist group,ROCK agonist + LIMK inhibitor group and DMSO control group.The detection of osteoblasts proliferation,migration and osteogenic differentiation included the following experiments: CCK-8method was used to detect the changes of cell proliferation,then cell migration and Transwell were used to detect the changes of cell migration.In the second part,the changes of osteoblast cytoskeleton and osteogenic ability were detected by the following experiments: the level of Runx2 and cytoskeleton detected by immunofluorescence,and the expressions of pathway proteins and osteogenic related proteins ROCK,LIMK,p-LIMK,cofilin,p-cofilin and Runx2 were detected by Western blot.Then,the cells were divided into four groups before osteogenic induction: ROCK agonist group,ROCK agonist + LIMK inhibitor group,DMSO control group,and DMSO control group without osteogenic induction solution.After3 days of induction,the osteogenic ability of the cells was detected as follows.The expression of osteogenic related m RNA: Runx2,ALP were detected by real-time PCR,alkaline phosphatase staining test and alkaline phosphatase activity test were used to detect the osteogenic ability of the osteoblasts.At last,in vivo experiment,we placed a force device in the cranial suture of mice for distraction with or without ROCK inhibitor to change the expression of ROCK.Then we measured the distraction distance by direct measurement and HE staining.We used the Western blot to detect the protein changes of pathway and Runx2.Results: Activation of ROCK can affect the migration and osteogenic differentiation of 3T3-E1 osteoblasts through ROCK-LIMK-cofilin axis,but has no effect on the early proliferation of osteoblasts.Activation of ROCK can increase the expression of downstream cytoskeleton related proteins p-LIMK,p-cofilin and osteogenic related protein Runx2,thus promoting actin contraction and enhancing osteoblast osteogenesis and differentiation migration ability.The addition of LIMK inhibitor could inhibit the expression of p-LIMK,p-cofilin and Runx2,but not the total amount of LIMK and cofilin,thus promoting the depolymerization of actin and reducing the osteogenic and migration ability of osteoblasts.In addition,the addition of LIMK inhibitor could inhibit the proliferation of osteoblasts from the fourth day.By inhibiting the expression of ROCK in vivo,we found that the reduction of new bone formation,and the expression of cytoskeleton related proteins and Runx2 were decreased.Conclusion: ROCK-LIMK-Cofilin pathway plays an important role in regulating the migration and osteogenic differentiation of osteoblasts.