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

Abstract

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 ␤ (C/EBP␤) message. To examine the function and importance of the HuR-C/EBP␤ interaction, retroviral expression constructs were created in which the HuR binding site was altered by deletion (␤del) or deletion and substitution (␤d/s). Expression of these constructs in murine embryonic fibroblasts resulted in significant adipose conversion relative to those cells expressing wild type C/EBP␤. C/EBP␤ protein content was increased markedly in both ␤del and ␤d/s, which correlated with the acquisition of the adipocyte phenotype. Analysis of the ␤d/s cell line demonstrated a robust expression of C/EBP␣ coincident with peroxisome proliferatoractivated receptor ␥ expression. Total C/EBP␤ mRNA accumulation indicated no difference between cells harboring either the wild type C/EBP␤ cDNA or ␤d/s construct. However, cytosolic C/EBP␤ mRNA in the cells expressing the ␤d/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/EBP␤ mRNA and is consistent with HuR control, at least in part, of mRNA processing.

Altered Regulation of ELAVL1/HuR in HLA-B27–Expressing U937 Monocytic Cells

“…HuR enhances C/EBPβ mRNA stability and translation, leading to increased amount of the protein [18]. Controversial findings where HuR binding decreases the C/EBPβ expression exist, HuR binding to C/EBPβ mRNA is suggested to diminish its movement to cytosol [22]. Thus it is also possible that the regulation of HuR binding is disturbed in HLA-B27 cells and this leads to increased cytoplasmic C/EBPβ mRNA and protein production and results in C/EBPβ overexpression.…”

“…This function appears to be regulated by caspase-dependent cleavage of HuR [13], [21]. HuR is mainly localized in the nucleus but can shuttle between nucleus and cytoplasm [22]. In cytoplasm, HuR can be cleaved to two cleavage products (CPs), HuR-CP1 (24 kDa) and HuR-CP2 (8 kDa), that have been linked to promotion of apoptosis [21].…”

HuR involvement in mitotic clonal expansion during acquisition of the adipocyte phenotype

“…Briefly, cells were transfected twice, the first time at 60% confluence, and the second 24h later while the cells remained pre-confluent. At 24h after the second transfection, two monolayers were combined to generate immediate confluency, 24h after the second transfection differentiation was initiated as we have previously described [3,7]. The 0 time point represents the time of exposure of the cells to the differentiation inducers.…”

“…Protein extracts were prepared as previously described [3,7] and Western blot analysis performed as previously detailed [3,7]. …”

“…Whether the function of the complex is export from the nucleus or stabilization of the message, disruption of the interaction leads to alteration of expression and attenuation of the differentiation program. In terms of timing, formation of the HuR-mRNA complex has to be early in the process, because as we have previously demonstrated disruption of this interaction must precede expression of C/EBPβ [7]. In addition, more than one ligand mRNA may be involved in the process.…”