Posttranscriptional regulation of chicken ccn2 gene expression by nucleophosmin/B23 during chondrocyte differentiation

Biodental Research Center, Okayama University Dental School, Okayama 700-8525, Japan.
Molecular and Cellular Biology (Impact Factor: 5.04). 09/2008; 28(19):6134-47. DOI: 10.1128/MCB.00495-08
Source: PubMed

ABSTRACT CCN2/CTGF is a multifunctional factor that plays a crucial role in the growth and differentiation of chondrocytes. The chicken ccn2 gene is regulated not only at the transcriptional level but also by the interaction between a posttranscriptional element in the 3' untranslated region (3'-UTR) and a cofactor. In the present study, we identified a nucleophosmin (NPM) (also called B23) as this cofactor. Binding of NPM to the element was confirmed, and subsequent analysis revealed a significant correlation between the decrease in cytosolic NPM and the increased stability of the ccn2 mRNA during chondrocyte differentiation in vivo. Furthermore, recombinant chicken NPM enhanced the degradation of chimeric RNAs containing the posttranscriptional cis elements in a chicken embryonic fibroblast extract in vitro. It is noteworthy that the RNA destabilization effect by NPM was far more prominent in the cytosolic extract of chondrocytes than in that of fibroblasts, representing a chondrocyte-specific action of NPM. Stimulation by growth factors to promote differentiation changed the subcellular distribution of NPM in chondrocytes, which followed the expected patterns from the resultant change in the ccn2 mRNA stability. Therefore, the present study reveals a novel aspect of NPM as a key player in the posttranscriptional regulation of ccn2 mRNA during the differentiation of chondrocytes.

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Available from: Seiji Kondo, Aug 25, 2015
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    • "For example, in another CCN family member, CCN2, cis-acting element of structure-anchored repression was discovered [Kondo et al., 2000; Kubota et al., 2000, 2005]. Furthermore, our group recently clarified that chicken ccn2 mRNA level is regulated by selective mRNA degradation under the collaboration of a structured mRNA element (5 0 –100/50) and nucleophosmin/B23 [Mukudai et al., 2008]. "
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    ABSTRACT: CCN1, a member of the CCN family of proteins, plays important physiological or pathological roles in a variety of tissues. In the present study, we initially found a highly guanine-cytosine (GC)-rich region of approximately 200 bp near the 5'-end of the open reading frame, which was always truncated by amplification of the corresponding cDNA region through the conventional polymerase chain reaction. An RNA in vitro folding assay and selective ribonuclease digestion of the corresponding segment of the ccn1 mRNA confirmed the involvement of a stable secondary structure. Subsequent RNA electromobility-shift assays demonstrated the specific binding of some cytoplasmic factor(s) in chicken embryo fibroblasts to the RNA segment. Moreover, the corresponding cDNA fragment strongly enhanced the expression of the reporter gene in cis at the 5'-end, but did not do so at the 3'-end. According to the results of a ribosomal assembly test, the effect of the mRNA segment can predominantly be ascribed to the enhancement of transport and/or entry of the mRNA into the ribosome. Finally, the minimal GC-rich mRNA segment that was predicted and demonstrated to form a secondary structure was confirmed to be a functional regulatory element. Thus, we here uncover a novel dual-functionality of the mRNA segment in the ccn1 open reading frame, which segment acts as a cis-element that mediates posttranscriptional gene regulation, while retaining the information for the amino acid sequence of the resultant protein.
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    • "Long open box denotes the UTR flanked by the coding sequence (CDS: upstream) and poly (A) tail (downstream). Cis-repressive RNA elements that were previously identified and designated ''CAESAR " [8] and ''3 0 -100/50 " [9] are also displayed in rectangular frames with their names. Solid and open small boxes indicate evolutionally conserved and non-conserved sequences, respectively, which were predicted to be the targets of the corresponding miRNAs. "
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    ABSTRACT: We searched for miRNAs that were down-regulated in chondrocytic cells and predicted to target CCN2/connective tissue growth factor (CCN2/CTGF) that promotes endochondral ossification. Among them, expression of miR-18a was most strongly repressed in chondrocytic cells. Reporter gene analysis confirmed the functionality of an miR-18a target in the 3'-untranslated region of Ccn2 mRNA, which was predicted in silico. Indeed, introduction of miR-18a efficiently repressed the CCN2 production from chondrocytic cells. Finally, transfected miR-18a significantly repressed the mature chondrocytic phenotype. Our present study revealed a regulatory role for miR-18a in chondrocytic differentiation through CCN2.
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    ABSTRACT: CCN2 (connective tissue growth factor (CTGF)/hypertrophic chondrocyte-specific gene product 24 (Hcs24)) is regulated at the transcriptional and posttranscriptional level. For example, an element in the its 3'untranslated region (3'-UTR) of the CCN2 mRNA controls message stability in chondrocytes. In a recent study, Mukudai and colleagues (2008) purified and identified a trans- factor protein binding to the minimal repressive cis element in the 3'-UTR of ccn2 mRNA and identify this protein as the multifunctional nucleolar phosphoprotein nucleophosmin (NPM) This commentary summarizes these observations.
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