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Study of the DNA Damage Checkpoint using Xenopus Egg Extracts
Published on: November 5, 2012
DNA damage checkpoint pathway modulates the regulation of skeletal growth and osteoblastic bone formation by
Ying Zhang1,2, Guangpei Chen1, Zhen Gu1
1State Key Laboratory of Reproductive Medicine, The Research Center for Bone and Stem Cells, Department of Anatomy, Histology and Embryology, Nanjing Medical University, Nanjing, China.
Abstract:
We previously demonstrated that parathyroid hormone-related peptide (PTHrP) 1-84 knockin (Pthrp KI) mice, which lacked a PTHrP nuclear localization sequence (NLS) and C-terminus, displayed early senescence, defective osteoblastic bone formation, and skeletal growth retardation. However, the mechanism of action of the PTHrP NLS and C-terminus in regulating development of skeleton is still unclear. In this study, we examined alterations of oxidative stress and DNA damage response-related molecules in Pthrp KI skeletal tissue. We found that ROS levels, protein expression levels of γ-H2AX, a DNA damage marker, and the DNA damage response markers p-Chk2 and p53 were up-regulated, whereas gene expression levels of anti-oxidative enzymes were down-regulated significantly. We therefore further disrupted the DNA damage response pathway by deleting the Chk2 in Pthrp KI (Chk2-/-KI) mice and did comparison with WT, Chk2-/- and Pthrp KI littermates. The Pthrp KI mice with Chk2 deletion exhibited a longer lifespan, improvement in osteoblastic bone formation and skeletal growth including width of growth plates and length of long bones, trabecular and epiphyseal bone volume, BMD, osteoblast numbers, type I collagen and ALP positive bone areas, the numbers of total colony-forming unit fibroblasts (CFU-f), ALP+ CFU-f and the expression levels of osteogenic genes. In addition, the genes associated with anti-oxidative enzymes were up-regulated significantly, whereas the tumor suppressor genes related to senescence were down-regulated in Chk2 KI mice compared to Pthrp KI mice. Our results suggest that Chk2 deletion in Pthrp KI mice can somewhat rescue defects in osteoblastic bone formation and skeletal growth by enhancing endochondral bone formation and osteogenesis. These studies therefore indicate that the DNA damage checkpoint pathway may be a target for the nuclear action of PTHrP to regulate skeletal development and growth.
Insights
Parathyroid hormone-related peptide (PTHrP) deficiency causes skeletal defects. Deleting Chk2 in these mice rescues bone formation and growth by reducing DNA damage and oxidative stress.
Area of Science:
- Molecular Biology
- Skeletal Biology
- Genetics
Background:
- Parathyroid hormone-related peptide (PTHrP) is crucial for skeletal development.
- PTHrP's nuclear localization sequence (NLS) and C-terminus roles in skeletal regulation are unclear.
- PTHrP knockin (KI) mice lacking NLS/C-terminus show senescence and growth defects.
Purpose of the Study:
- Investigate the mechanism of PTHrP NLS/C-terminus in skeletal development.
- Examine oxidative stress and DNA damage response in PTHrP KI mice.
- Determine if disrupting the DNA damage response pathway can rescue skeletal defects.
Main Methods:
- Analyzed oxidative stress and DNA damage markers (ROS, γ-H2AX, p-Chk2, p53) in PTHrP KI skeletal tissue.
- Generated and compared PTHrP KI mice with Chk2 deletion (Chk2-/-KI) to wild-type (WT) and other controls.
- Assessed skeletal growth, bone formation, cell numbers, and gene expression in different mouse models.
Main Results:
- PTHrP KI mice exhibited increased ROS, DNA damage markers, and decreased anti-oxidative enzyme expression.
- Chk2 deletion in PTHrP KI mice (Chk2-/-KI) improved lifespan, bone formation, and skeletal growth.
- Chk2 deletion normalized anti-oxidative enzyme expression and reduced senescence-related genes compared to PTHrP KI mice.
Conclusions:
- The DNA damage response pathway, particularly Chk2, is implicated in PTHrP's regulation of skeletal development.
- Disrupting the Chk2 pathway rescues osteoblastic bone formation and skeletal growth defects in PTHrP KI mice.
- Targeting the DNA damage checkpoint pathway offers a potential therapeutic strategy for skeletal disorders related to PTHrP.
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