2014 Fiscal Year Research-status Report
Pathophysiological investigation of bone cell communications regulated by chemokine network
| Project/Area Number |
26861549
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| Research Institution | Ehime University |
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Principal Investigator |
李 智媛 愛媛大学, プロテオサイエンスセンター, 助教(特命教員) (70711274)
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| Project Period (FY) |
2014-04-01 – 2016-03-31
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| Keywords | chemokine / osteoclast / bone / small GTPase |
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| Outline of Annual Research Achievements |
-Impairment of bone resorption function in osteoclasts from Ccr5-deficiency mice, not osteoblast function; Osteoclastic cells isolated from bone marrow of wild-type and Ccr5-deficient mice were showed no siginificant in cell proliferation and differentiation. But, live imaging and super-resolution microscopy analysis revealed that Ccr5-deificient osteoclasts showed larger in size and disorganized motility, cytoskeletal rearrangent and cell-atttachment machineries. Its mechanism action is specifically involved the cytoskeletal organization of mature osteoclasts associated with Src via small GTPase activation, finally leading to reduced osteoclast function.
-Increased bone mass due to the decreased function of osteoclastic bone resorption in Ccr5-deficiency mice; We analyzed femur bones from 10 week-old Ccr5-deficiency and wild-type mice by microCT and administered calcein to evaluate osteoblast activity. Both bone formation parameters, as well as other serum marker (osteocalcin and trap) were not significantly different between the two genotypes. To further evaluate the affect by which CCR5 interferes with bone mass, we performed in vivo experiment using an sRANKL-induced bone loss model. MicroCT and bone morphometric analysis clearly showed that Ccr5-deficient mice exerted significant increases in bone mass as a result of a decrease in bone resorption in RANKL-induced bone loss model, suggesting functional impairment of osteoclasts.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The 1st year of project is being under processing as planed.
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| Strategy for Future Research Activity |
-Recover experiment of osteoclastic function and mechanism elucidation of intracellular signal; Since we found that the expression level of osteoclastic markers and adhesive markers such as integrin, mmp3, mmp13 and small GTPases, were significantly decreased compare to wild-type and Ccr5-deficiency mice, it is important to investigate integrin and chemokine signal network and further whether these internal and external signal can recover to control level. For this, we will perform the transfection of Small GTPases, Rac, Rho or Cdc42 etc. using adeno-virus system and evaluation of bone resorption (pit formation and actin ring formation) finally.
-Analysis of roles of CCR5 in human osteoclasts and osteoblasts in vitro; We will observe the bio-markers of osteoclasts by quantitative RT-PCR, osteoclast differentiation and function, applying with human anti-CCR5 neutralizing antibody. And it is also evaluate osteoblast differentiation and mineralization from human osteoblastic cells.
-Functional dissection of CCR5 from CX3CR1 and CCR1 and their inter-regulations in osteoclasts; Previous studies by our group, CX3CR1 and CCR1 play critical roles in osteoclast precursors and early differentiated osteoclasts, respectively. Based our CCR5 results, functional analyses of these three chemokine receptors in osteoclasts will help to retrieve functional redundancies and distinctions. We would like to investigate chemokines and their cell migration including cytoskeletal arrangement and cell attachment, and cellular interaction of osteoclasts in vitro.
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