An Early Event in Adipogenesis, the Nuclear Selection of the CCAAT Enhancer-binding Protein β (C/EBPβ) mRNA by HuR and its Translocation to the Cytosol

Abstract

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-Mediated Control of C/EBPβ mRNA Stability and Translation in ALK-Positive Anaplastic Large Cell Lymphomas

“…On the other hand, C/EBPb, has been intensely studied for its critical role in adipocyte differentiation. During this process, C/EBPb gene expression has been shown to be regulated at the posttranscriptional level by a mechanism involving the formation of a nuclear HuR/C/EBPb mRNA complex that determines the amount of message reaching the cytosol (23,24). In this study, we have established a link between these different analyses and have shown that in ALK þ ALCLs, NPM-ALK exerts a posttranscriptional control on C/EBPb gene expression that implicates HuR, and leads to increased C/EBPb mRNA stability and translation.…”

Post-transcriptional regulation in metabolic diseases

“…Gantt and colleagues showed that HuR plays a role during acquisition of the adipocyte phenotype. 71 HuR was constitutively expressed and localized predominantly to the nucleus in the preadipocytes, whereas adipogenic stimulus increased the HuR content in the cytosol and rapid formation of HuR complexes containing C/EBPβ mRNA. In the 3T3-L1 cell, suppression of HuR expression resulted in a decrease of C/EBPβ expression and accumulation of lipid droplets and, in turn, an inhibition of adipogenesis, suggesting that HuR functions in establishing the adipocyte phenotype.…”

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

“…In 3T3-L1 preadipocytes, HuR is constitutively expressed and localized predominantly to the nucleus (28). 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.…”

“…Isolation of Nuclear and Cytosolic Fractions-The NE-PER TM cell fractionation kit was used to isolate nuclear and cytosolic fractions from the cells following the manufacturer's instructions with minor modifications as described previously (28). For isolation of RNA, 4 wells of a 6-well cluster plate were used as a source of material for the separation.…”

“…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. We have demonstrated previously (28) that one of the relevant mRNAs forming a nuclear complex with HuR upon induction of the differentiation program and translocating to the cytosol as a messenger ribonucleoprotein (mRNP) complex with HuR is the C/EBP␤ message.…”