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Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow
Published on: July 15, 2016
Cytosolic Carboxypeptidase 1 Loss in Bone Marrow Mesenchymal Stem Cells Inhibited Osteogenic Differentiation by
Cancan Pan1, Xuyan Gong1, Xuekui Wang1
1Shanghai Engineering Research Center of Tooth Restoration and Regeneration and Tongji Research Institute of Stomatology and Department of Oral Implantology, Shanghai Tongji Stomatological Hospital and Dental School, Tongji University, Shanghai, China.
Introduction:
Cytosolic carboxypeptidase 1 (CCP1) is a deglutamylase that antagonizes polyglutamylation. Mutations in human CCP1 gene cause a severe disease known as childhood-onset neurodegeneration with cerebellar atrophy (CONDCA), which is characterized by marked growth retardation. However, the role and mechanisms of CCP1 in skeletal development remain unclear.
Methods:
In this study, we used CCP1 knockout (CCP1-KO) mice to assess bone mass changes by micro-CT, HE, alkaline phosphatase (ALP) staining, tartrate-resistant acid phosphatase staining and immunofluorescence staining. Changes in osteogenic differentiation, proliferation, and migration capacity of bone marrow mesenchymal stem cells (BMSCs) were assessed by ALP, alizarin red (ARS) staining, quantitative real-time PCR, EdU staining, and cell scratching assay. Then, tubulin glutamylation and primary cilia of BMSCs after deletion of CCP1 was analyzed by Western blot and immunofluorescence staining. Finally, CB839, an inhibitor of glutamine metabolism, was used to detect changes in the osteogenic differentiation ability and primary cilia of BMSCs after reducing the elevated glutamylation level.
Results:
CCP1-KO mice exhibited phenotypes relevant to humans, including reduced body size, decreased bone mass, and reduced bone density during growth and development. CCP1 deficiency impairs the proliferation, migration, and osteogenic differentiation of BMSCs. Meanwhile, the number of pre-osteoblasts derived from BMSCs is decreased, leading to impaired osteogenesis. At the cellular level, CCP1 loss results in aberrant tubulin glutamylation, increased microtubule glutamylation, and shortened primary cilia in BMSCs. Finally, reduction of abnormally elevated tubulin glutamylation was efficacious for promoting osteogenic differentiation of BMSCs and restoring primary cilia length of BMSCs.
Conclusion:
We propose that CCP1 plays a critical role in regulating BMSCs differentiation and promotes osteogenesis by modulating the post-translational modifications of tubulin, with a view to provide new targets for the prevention and treatment of hard tissue diseases.
Insights
Cytosolic carboxypeptidase 1 (CCP1) deficiency impairs bone development and mesenchymal stem cell differentiation by altering tubulin glutamylation. Restoring tubulin glutamylation promotes osteogenesis and primary cilia length, suggesting CCP1 as a therapeutic target for bone diseases.
Area of Science:
- Biochemistry
- Cell Biology
- Developmental Biology
Background:
- Cytosolic carboxypeptidase 1 (CCP1) antagonizes polyglutamylation and its mutations cause childhood-onset neurodegeneration with cerebellar atrophy (CONDCA).
- The role of CCP1 in skeletal development and its underlying mechanisms remain largely unknown.
Purpose of the Study:
- To investigate the function of CCP1 in skeletal development and bone marrow mesenchymal stem cell (BMSC) differentiation.
- To elucidate the molecular mechanisms by which CCP1 influences osteogenesis, focusing on tubulin glutamylation and primary cilia.
Main Methods:
- Utilized CCP1 knockout (CCP1-KO) mice for phenotypic analysis (micro-CT, histology).
- Assessed BMSC proliferation, migration, and osteogenic differentiation using various in vitro assays (ALP, ARS, qRT-PCR, EdU, cell scratching).
- Analyzed tubulin glutamylation and primary cilia in BMSCs via Western blot and immunofluorescence; utilized CB839 (glutamine metabolism inhibitor) to modulate glutamylation levels.
Main Results:
- CCP1-KO mice displayed reduced body size, bone mass, and bone density.
- CCP1 deficiency impaired BMSC proliferation, migration, and osteogenic differentiation, decreasing pre-osteoblast numbers.
- CCP1 loss led to aberrant tubulin glutamylation, increased microtubule glutamylation, and shortened primary cilia in BMSCs.
- Reducing elevated tubulin glutamylation restored BMSC osteogenic differentiation and primary cilia length.
Conclusions:
- CCP1 is crucial for skeletal development and osteogenesis by regulating BMSC differentiation.
- CCP1 modulates tubulin post-translational modifications (PTM), impacting microtubule function and primary cilia.
- Targeting tubulin glutamylation offers a potential therapeutic strategy for hard tissue diseases associated with CCP1 dysfunction.
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