Thrombin activates a Y box-binding protein (DNA-binding protein B) in endothelial cells

Area of Science:

• Molecular Biology
• Cell Signaling
• Gene Regulation

Background:

• Thrombin is known to stimulate gene expression in endothelial cells (ECs).
• The specific trans-acting factors mediating thrombin-induced gene expression in ECs were previously undefined.
• A thrombin-inducible nuclear factor (TINF) was previously identified, binding to the PDGF B promoter.

Purpose of the Study:

• To identify and characterize the trans-acting factors responsible for thrombin-induced gene expression in endothelial cells.
• To elucidate the mechanism of TINF activation and its role in PDGF B gene regulation.

Main Methods:

• Purification of TINF from thrombin-treated ECs.
• Amino acid sequencing and cDNA analysis to identify TINF.
• Expression of GFP-dbpB fusion proteins and FACS analysis to track nuclear translocation.
• In vitro assays with protein tyrosine phosphatase inhibitors to assess the role of phosphatases in dbpB activation.

Main Results:

• TINF was identified as DNA-binding protein B (dbpB), a member of the Y-box protein family.
• Thrombin stimulation caused dbpB to translocate to the nucleus in ECs.
• Thrombin activation resulted in the cleavage of dbpB, generating a fragment that binds the thrombin-response element but not the Y-box consensus sequence.
• Inhibition of protein tyrosine phosphatases blocked dbpB activation and thrombin-induced PDGF B-chain gene expression.

Conclusions:

• DNA-binding protein B (dbpB) is a key mediator of thrombin-induced gene expression in endothelial cells.
• Thrombin activates dbpB through a novel signaling pathway involving protein tyrosine phosphatases and proteolytic cleavage.
• This study reveals a new mechanism for regulating gene expression by activating Y-box proteins in response to agonists.