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Protein tyrosine phosphatase regulation of endothelial cell apoptosis and differentiation
1Laboratory of Biological Chemistry, National Institute on Aging, Baltimore, Maryland 21224, USA.
Abstract:
Apoptosis, or programmed cell death, occurs during development and may also be an important factor in many diseases. However, little is known about the signal transduction pathways regulating apoptosis. In these studies, loss of endothelial cell-substrate attachment and apoptosis after removal of growth factors was associated with dephosphorylation of tyrosine residues at the cell periphery. Dephosphorylation of total cellular proteins accompanied apoptosis and was reduced by orthovanadate, an inhibitor of protein tyrosine phosphatases. Orthovanadate blocked the fragmentation of nuclear DNA, inhibited DNA laddering, and suppressed the expression of TRPM-2, an apoptosis-associated gene. The tyrosine phosphorylation levels of FAK125, erk1 (mitogen-activated kinase kinase), and cdc-2 were reduced during apoptosis. FAK125 dephosphorylation was inhibited by orthovanadate, but premature activation (tyrosine dephosphorylation) of cdc-2 was not. Orthovanadate was as effective as basic fibroblast growth factor in activating erk1 without increasing cell proliferation and in preventing the apoptosis of endothelial cells after treatment with tumor necrosis factor alpha. Endothelial cell differentiation on extracellular matrix (Matrigel) was also stimulated by orthovanadate in the absence of basic fibroblast growth factor without affecting growth arrest and inhibition of DNA synthesis. Expression of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1) was down-regulated during the early stages of differentiation, remained low for at least 6 hours as differentiation proceeded, and increased upon completion of differentiation. Cells that failed to down-regulate p21 mRNA on Matrigel in the absence of angiogenic factors underwent apoptosis. These results suggest that protein tyrosine phosphatases are actively involved in signal transduction during apoptosis and may regulate p21 expression to inhibit endothelial cell differentiation.
Insights
Protein tyrosine phosphatases regulate programmed cell death (apoptosis) by influencing cell signaling pathways. Inhibiting these phosphatases with orthovanadate prevents DNA fragmentation and promotes endothelial cell differentiation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Apoptosis, or programmed cell death, is crucial in development and disease, yet its regulatory signal transduction pathways remain poorly understood.
- Endothelial cell apoptosis after growth factor withdrawal involves peripheral tyrosine dephosphorylation.
Purpose of the Study:
- To investigate the role of protein tyrosine phosphatases in regulating endothelial cell apoptosis and differentiation.
- To identify specific signaling molecules involved in these processes.
Main Methods:
- Utilized orthovanadate, a protein tyrosine phosphatase inhibitor, to study its effects on endothelial cells.
- Assessed apoptosis markers, including DNA fragmentation and TRPM-2 gene expression.
- Analyzed tyrosine phosphorylation levels of FAK125, erk1, and cdc-2.
- Examined the expression of p21 (Waf1/Cip1/Sdi1) during differentiation and apoptosis.
Main Results:
- Orthovanadate inhibited apoptosis, DNA fragmentation, and TRPM-2 expression.
- Apoptosis was associated with decreased tyrosine phosphorylation of FAK125, erk1, and cdc-2.
- Orthovanadate promoted endothelial cell differentiation on Matrigel and activated erk1 without affecting proliferation.
- Down-regulation of p21 mRNA was essential for differentiation; failure to do so led to apoptosis.
Conclusions:
- Protein tyrosine phosphatases are actively involved in apoptosis signal transduction.
- These phosphatases may regulate p21 expression, thereby controlling endothelial cell differentiation and preventing apoptosis.