OM (oncostatin M) activates the human LDLR [LDL (low-density lipoprotein) receptor] gene transcription in HepG2 cells through the SIRE (sterol-independent regulatory element) of LDLR promoter. The SIRE sequence consists of a C/EBP (CCAAT/enhancer-binding protein)-binding site and a CRE (cAMP-response element). Our previous studies [Zhang, Ahlborn, Li, Kraemer and Liu (2002) J. Lipid Res. 43, 1477-1485; Zhang, Lin, Abidi, Thiel and Liu (2003) J. Biol. Chem. 278, 44246-44254] have demonstrated that OM transiently induces EGR-1 (early growth response gene product 1) expression and EGR-1 activates LDLR transcription primarily through a protein-protein interaction with C/EBPbeta, which serves as a co-activator of EGR-1. In the present study, we examined the direct role of C/EBPbeta as a transactivator in OM-regulated LDLR gene transcription independent of EGR-1. We show that OM induces C/EBPbeta expression with kinetics slower than EGR-1 induction. A significant increase in C/EBPbeta protein level is detected by 2 h of OM treatment and remains elevated for 24 h. Chromatin immunoprecipitation assays demonstrate that the amount of C/EBPbeta bound to the LDLR SIRE sequence is increased 2.8-fold of control by 2 h of OM treatment, reached the highest level of 8-fold by 4 h, and slowly declined thereafter. To further examine the requirement of C/EBPbeta in OM-stimulated LDLR expression, we developed a His-tagged dominant-negative mutant of C/EBPbeta (His-C/EBPbeta-P4; where P4 is plasmid 4 in our mutation series), consisting of the DNA-binding and leucine zipper domains of C/EBPbeta (amino acids 246-345). Expression of His-C/EBPbeta-P4 in HepG2 cells significantly diminishes the OM-induced increase in LDLR promoter activity and the elevation of endogenous LDLR mRNA expression. Taken together, these new findings identify C/EBPbeta as an OM-induced transactivator in LDLR gene transcription and provide a better understanding of the molecular mechanism underlying the sterol-independent regulation of LDLR expression.