Post-transcriptional Control of CCAAT/Enhancer-binding Protein β (C/EBPβ) Expression

In 3T3-L1 cells, HuR is constitutively expressed and prior to induction of differentiation localized predominantly to the nucleus. Within minutes of induction of differentiation, nuclear HuR binds to its target ligand mRNAs, and the complexes appear to move to the cytosol. One ligand mRNA is the CCAAT/enhancer-binding protein β (C/EBPβ) message. To examine the function and importance of the HuR-C/EBPβ interaction, retroviral expression constructs were created in which the HuR binding site was altered by deletion (βdel) or deletion and substitution (βd/s). Expression of these constructs in murine embryonic fibroblasts resulted in significant adipose conversion relative to those cells expressing wild type C/EBPβ. C/EBPβ protein content was increased markedly in both βdel and βd/s, which correlated with the acquisition of the adipocyte phenotype. Analysis of the βd/s cell line demonstrated a robust expression of C/EBPα coincident with peroxisome proliferator-activated receptor γ expression. Total C/EBPβ mRNA accumulation indicated no difference between cells harboring either the wild type C/EBPβ cDNA or βd/s construct. However, cytosolic C/EBPβ mRNA in the cells expressing the βd/s construct was maintained at levels between 2- and 7-fold greater than in the cells expressing the wild type construct. Alteration in mRNA half-life was not responsible for the increased accumulation. Mechanistically, these data suggest that HuR binding results in nuclear retention of the C/EBPβ mRNA and is consistent with HuR control, at least in part, of mRNA processing. In 3T3-L1 cells, HuR is constitutively expressed and prior to induction of differentiation localized predominantly to the nucleus. Within minutes of induction of differentiation, nuclear HuR binds to its target ligand mRNAs, and the complexes appear to move to the cytosol. One ligand mRNA is the CCAAT/enhancer-binding protein β (C/EBPβ) message. To examine the function and importance of the HuR-C/EBPβ interaction, retroviral expression constructs were created in which the HuR binding site was altered by deletion (βdel) or deletion and substitution (βd/s). Expression of these constructs in murine embryonic fibroblasts resulted in significant adipose conversion relative to those cells expressing wild type C/EBPβ. C/EBPβ protein content was increased markedly in both βdel and βd/s, which correlated with the acquisition of the adipocyte phenotype. Analysis of the βd/s cell line demonstrated a robust expression of C/EBPα coincident with peroxisome proliferator-activated receptor γ expression. Total C/EBPβ mRNA accumulation indicated no difference between cells harboring either the wild type C/EBPβ cDNA or βd/s construct. However, cytosolic C/EBPβ mRNA in the cells expressing the βd/s construct was maintained at levels between 2- and 7-fold greater than in the cells expressing the wild type construct. Alteration in mRNA half-life was not responsible for the increased accumulation. Mechanistically, these data suggest that HuR binding results in nuclear retention of the C/EBPβ mRNA and is consistent with HuR control, at least in part, of mRNA processing.