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Cell-cycle dependent phosphorylation of HSP28 by TGF beta 1 and H2O2 in normal mouse osteoblastic cells (MC3T3-E1),
M Shibanuma1, T Kuroki, K Nose
1Department of Cancer Cell Research, University of Tokyo, Japan.
Abstract:
Transforming growth factor (TGF) beta 1 increased phosphorylation of a specific protein of approximately M(r) = 30,000 (p30) in mouse osteoblastic MC3T3-E1 cells. This protein, p30, was identified as one of the small heat shock proteins (HSP) 28 from the electrophoretic pattern on two-dimensional gels, and its peptide map compared with that of heat shock-inducible p28. The increase in phosphorylation of HSP 28 seemed to correlate with growth inhibition in this cell line, since it was increased by growth inhibitory agents, such as TGF beta 1, H2O2 and 12-O-tetradecanoylphorbol-13-acetate (TPA), but not by the growth stimulating agent, epidermal growth factor (EGF), and this phosphorylation was observed only when the cells were sensitive to growth inhibition by these agents, in the late G1 phase of the cell cycle. Furthermore, in ras-transformants, whose DNA synthesis was not inhibited by these agents, this phosphorylation was not increased by these stimuli. These results indicate that phosphorylation of HSP 28 may be coupled to inhibition of DNA synthesis in this cell line.
Insights
Transforming growth factor-beta 1 (TGF-β1) increases the phosphorylation of heat shock protein 28 (HSP 28) in osteoblastic cells. This modification correlates with growth inhibition, suggesting HSP 28
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Osteoblastic cells play a crucial role in bone metabolism.
- Growth factors and cellular stress can modulate cell proliferation and differentiation.
- Heat shock proteins (HSPs) are involved in cellular stress responses and protein homeostasis.
Purpose of the Study:
- To investigate the effect of transforming growth factor-beta 1 (TGF-β1) on protein phosphorylation in osteoblastic cells.
- To identify the specific protein whose phosphorylation is altered by TGF-β1.
- To determine the relationship between protein phosphorylation and cell growth inhibition.
Main Methods:
- Two-dimensional gel electrophoresis to analyze protein phosphorylation.
- Peptide mapping to identify phosphorylated proteins.
- Cell cycle analysis to assess sensitivity to growth inhibition.
- Treatment with various agents including TGF-β1, H2O2, 12-O-tetradecanoylphorbol-13-acetate (TPA), and epidermal growth factor (EGF).
Main Results:
- TGF-β1 increased the phosphorylation of a ~30,000 M(r) protein (p30) in MC3T3-E1 cells.
- p30 was identified as heat shock protein 28 (HSP 28).
- HSP 28 phosphorylation correlated with growth inhibition induced by TGF-β1, H2O2, and TPA, particularly in the late G1 phase.
- This phosphorylation was not observed in ras-transformed cells resistant to growth inhibition.
- Epidermal growth factor (EGF) did not induce HSP 28 phosphorylation.
Conclusions:
- Phosphorylation of HSP 28 is induced by growth inhibitory agents in osteoblastic cells.
- HSP 28 phosphorylation appears to be coupled with the inhibition of DNA synthesis.
- These findings suggest a role for HSP 28 phosphorylation in regulating cell proliferation in response to specific stimuli.