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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Effect of tyrosine phosphatase inhibitors on activation of dbpB and PDGF...
Effect of tyrosine phosphatase inhibitors on activation of dbpB and PDGF B–chain transcription. (a) Effect of sodium vanadate (Na3VO4) and sodium fluoride (NaF) on dbpB activation by thrombin. ECs were pretreated with Na3VO4 or NaF (400 μM for 30 minutes) and then stimulated with thrombin (10 U/mL for 2 hours). EMSA was performed as described in Methods. Control, extract from untreated EC; Thr, extract from EC stimulated with thrombin. (b) Effect of sodium vanadate (Na3VO4) and phenyl arsine oxide (PAO) on thrombin-induced PDGF B–chain mRNA in ECs. ECs were pretreated with Na3VO4 or PAO and then stimulated with thrombin (10 U/mL for 6 hours). Total RNA was extracted using Trizol reagent and Northern hybridization was performed using either the PDGF B–chain or GAPDH cDNAs as probe. (c) Effect of PTP inhibitors on thrombin-induced transcription driven by the PDGF B–chain promoter. Bovine ECs were transiently transfected (as described in Methods) with a luciferase reporter gene under the control of a 400-bp PDGF B–chain promoter fragment in pGL3-Basic vector (Promega Corp.). Pretreatment (30 minutes) with PTP inhibitors (400 μM Na3VO4, or 200 nM PAO) was initiated after a 6-hour transfection incubation. Lysates were prepared after a 15-hour incubation with thrombin (10 U/mL). Luciferase activity was normalized to β-galactosidase activity derived from cotransfected pSV β-galactosidase cDNA.