HuR is a ligand for nuclear mRNAs containing adenylate-uridylate-rich elements in the 3-untranslated region. Once bound to the mRNA, HuR is recognized by adapter proteins that then facilitate nuclear export of the complex. In the cytosol, HuR is thought to function to control stability and translation of its ligand message. In the 3T3-L1 cells HuR is constitutively expressed and localized predominantly to the nucleus in the preadipocytes. However, within 30 min of exposure to the differentiation stimulus the HuR content in the cytosol increases, consistent with HuR regulating the availability of relevant mRNAs for translation. Using in vitro RNA gel shifts, we have demonstrated that the CCAAT enhancer-binding protein ␤ (C/EBP␤) message is a ligand for HuR. Within 2 h of initiation of the differentiation process, HuR complexes containing C/EBP␤ mRNA could be isolated from the cytosolic compartment. Importantly, the process appears to be highly selective, as cyclin D1, which contains a putative HuR binding site and is expressed on the same time frame as C/EBP␤, was not found in the immunoprecipitated messenger ribonucleoprotein complexes. The proximity of this event to adipogenic stimuli and the importance of C/EBP␤ to the differentiation process have led us to hypothesize a role for HuR in the regulation of the onset of adipogenesis. In support of this hypothesis, small interfering RNA suppression of HuR protein content resulted in an inhibition of C/EBP␤ protein expression and an attenuation of the differentiation process.

HuR is a ligand for nuclear mRNAs containing adenylate-uridylate-rich (ARE) elements in the 3'-untranslated region. Once bound to the mRNA, HuR is recognized by adapter proteins that then facilitate nuclear export of the complex. In the cytosol, HuR is thought to function to control stability and translation of its ligand message. We have previously demonstrated that HuR is constitutively expressed in the 3T3-L1 cells and shuttles from the nucleus to the cytosol, but remains predominantly nuclear in the preadipocytes and that as the cells differentiate, there is a marked increase in the proportion of HuR in the cytosol at any time. The GLUT1 glucose transporter is also expressed in both preadipocytes and adipocytes and in vitro RNA gel shifts indicate the mRNA is a ligand for HuR. However, HuR complexes containing the GLUT1 mRNA can only be isolated from the terminally differentiated adipocytes. Moreover, position analysis of the GLUT1 mRNA and HuR protein in polysome profiles demonstrates a shift to the most dense region of the gradient for both message and protein with adipocyte differentiation. Consistent with a regulatory role in the control of GLUT1 expression, siRNA-mediated decrease in HuR protein resulted in a decreased expression of GLUT1 protein. These data suggest that HuR contributes to the metabolic function of the adipocyte through mediation of post-transcriptional regulatory events.

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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.

In the nucleus HuR binds to mRNAs containing adenylate-uridylate rich elements in the 3′-untranslated region. HuR may influence expression of its ligand mRNA through regulation of polyadenylation, translocation of the message to the cytosol, stabilization of the mRNA and/or altering its translational efficiency. Suppression of HuR using siRNA resulted in an attenuation of the 3T3-L1 differentiation program, consistent with HuR control of the expression of mRNA ligand (s) critical to the differentiation process. In the current study we begin to identify mRNA ligands of HuR whose regulated expression is necessary for adipogenesis.