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p57(Kip2) is degraded through the proteasome in osteoblasts stimulated to proliferation by transforming growth factor
T Urano1, H Yashiroda, M Muraoka
1Department of Tumor Biochemistry, Japan Science and Technology Corporation, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.
Abstract:
Cyclin-dependent kinase inhibitory proteins are negative regulators of the cell cycle. Although all the cyclin-dependent kinase inhibitory proteins may be involved in cell cycle control during a differentiation process, only p57(Kip2) is shown to be essential for embryonic development. However, the role of p57 in the control of the cell cycle is poorly understood. Using osteoblasts derived from the calvaria of rat fetus, we show that p57 is accumulated in cells starved by low serum. Cyclin-dependent kinase 2 activity was suppressed in these cells with a significant amount bound to p57. Treatment of the cells with transforming growth factor beta1 dramatically reduced the amount of p57, resulting in an activation of cyclin-dependent kinase 2 activity and the stimulation of cell proliferation. The decrease in p57 was inhibited by treating the cells with proteasome inhibitors, Z-Leu-Leu-Leu-aldehyde or lactacystin, but not with Z-Leu-Leu-aldehyde, which is an inhibitor of calpain, indicating that p57 is degraded through the proteasome pathway. p57 was also shown to be ubiquitinated in vitro. Because transforming growth factor beta1 not only stimulates the growth but also inhibits the differentiation of the cells in this system, our results may suggest a possible involvement of p57 in the control of osteoblastic cell proliferation and differentiation.
Insights
p57 Kip2 protein accumulation in osteoblasts suppresses cell cycle, while TGF-β1 reduces p57 Kip2, activating proliferation. This suggests p57 Kip2 controls osteoblastic cell growth and differentiation.
Area of Science:
- Cell Biology
- Molecular Biology
- Developmental Biology
Background:
- Cyclin-dependent kinase inhibitory proteins regulate the cell cycle.
- p57 Kip2 is crucial for embryonic development, but its role in cell cycle control is unclear.
- Osteoblasts are key bone-forming cells with critical cell cycle regulation.
Purpose of the Study:
- To investigate the role of p57 Kip2 in controlling osteoblastic cell proliferation and differentiation.
- To understand the regulation of p57 Kip2 levels and its impact on cell cycle activity.
Main Methods:
- Used rat fetal calvarial osteoblasts.
- Studied p57 Kip2 accumulation under low serum conditions.
- Assessed cyclin-dependent kinase 2 activity and its association with p57 Kip2.
- Investigated the effect of transforming growth factor beta1 (TGF-β1) on p57 Kip2 levels and cell proliferation.
- Utilized proteasome inhibitors (Z-Leu-Leu-Leu-aldehyde, lactacystin) and a calpain inhibitor (Z-Leu-Leu-aldehyde) to determine p57 Kip2 degradation pathways.
- Performed in vitro ubiquitination assays.
Main Results:
- p57 Kip2 accumulated in serum-starved osteoblasts, suppressing cyclin-dependent kinase 2 activity.
- TGF-β1 treatment decreased p57 Kip2 levels, activating cyclin-dependent kinase 2 and stimulating cell proliferation.
- Proteasome inhibitors blocked TGF-β1-induced p57 Kip2 degradation, indicating proteasomal degradation.
- p57 Kip2 was ubiquitinated in vitro, confirming its degradation pathway.
- TGF-β1 inhibited osteoblastic differentiation while promoting proliferation.
Conclusions:
- p57 Kip2 is degraded via the proteasome pathway in osteoblasts.
- TGF-β1-induced reduction of p57 Kip2 contributes to osteoblastic cell proliferation.
- p57 Kip2 may play a role in regulating both proliferation and differentiation of osteoblastic cells.