Thrombin activates a Y box–binding protein (DNA-binding protein B) in endothelial cells
Olga I. Stenina, Earl J. Poptic, Paul E. DiCorleto
Olga I. Stenina, Earl J. Poptic, Paul E. DiCorleto
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Thrombin activates a Y box–binding protein (DNA-binding protein B) in endothelial cells

Abstract

Thrombin stimulates the expression of multiple genes in endothelial cells (ECs), but the trans-acting factors responsible for this induction remain undefined. We have previously described a thrombin-inducible nuclear factor (TINF), which binds to an element in the PDGF B promoter and is responsible for the thrombin inducibility of this gene. Inactive cytoplasmic TINF is rapidly activated and translocated to nuclei of ECs upon stimulation with thrombin. We have now purified TINF from thrombin-treated ECs. Amino acid sequencing revealed it to be a member of the Y-box protein family, and the sole Y-box protein–encoding cDNA we detected in human or bovine ECs corresponded to DNA-binding protein B (dbpB). DbpB translocated to the nucleus after thrombin stimulation of ECs as shown by FACS analysis of nuclei from ECs expressing GFP-dbpB fusion proteins. During thrombin activation, dbpB was found to be cleaved, yielding a 30-kDa NH2-terminal fragment that recognized the thrombin-response element sequence, but not the Y-box consensus sequence. Preincubation of ECs with protein tyrosine phosphatase inhibitors completely blocked dbpB activation by thrombin and blocked induction of endogenous PDGF B–chain mRNA and promoter activation by thrombin. Y-box proteins are known to act constitutively to regulate the expression of several genes. Activation of this class of transcription factors in response to thrombin or any other agonist represents a novel signaling pathway.

Authors

Olga I. Stenina, Earl J. Poptic, Paul E. DiCorleto

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Comparison of truncated dbpB to TINF. A truncated dbpB cDNA was prepared...
Comparison of truncated dbpB to TINF. A truncated dbpB cDNA was prepared by introducing a stop codon following position 621 and subcloning into the pcDNA3 vector. The truncated dbpB cDNA was transcribed and translated in vitro and subjected to SDS-PAGE (8% gel) following cross-linking to the DNA probe (a) and EMSA (b). (The ThRE oligonucleotide was used as a probe in both assays). pcDNA3, empty pcDNA3 vector used as a control for the in vitro transcription and translation reaction; truncated dbpB, truncated dbpB cDNA in pcDNA3 vector used for an in vitro translation reaction; contro, extract from nonstimulated ECs; thrombin, extract from thrombin-stimulated ECs (10 U/mL for 2 hours).