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: 4.78). 09/2008; 28(19):6134-47. DOI: 10.1128/MCB.00495-08
Source: PubMed


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
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    • "The transient induction of ccn1/2/3 observed in this study could be necessary and essential in initiation of MSC differentiation. A cis-acting element of structure-anchored post-transcriptional repression (CAESAR) was identified in the 3′-UTR of ccn2 in human [59], [60], [61], then similar elements was found in mouse [60] and in chicken [62], [63]. In addition, miR-26ab/1297, miR-132/212, miR-133, miR-18ab and miR-19, members of OstemiR, were predicted to recognize the 3′-UTR of ccn2/ctgf. "
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    ABSTRACT: MicroRNAs (miRNAs) are small RNA molecules of 21-25 nucleotides that regulate cell behavior through inhibition of translation from mRNA to protein, promotion of mRNA degradation and control of gene transcription. In this study, we investigated the miRNA expression signatures of cell cultures undergoing osteoblastic and osteocytic differentiation from mesenchymal stem cells (MSC) using mouse MSC line KUSA-A1 and human MSCs. Ninety types of miRNA were quantified during osteoblastic/osteocytic differentiation in KUSA-A1 cells utilizing miRNA PCR arrays. Coincidently with mRNA induction of the osteoblastic and osteocytic markers, the expression levels of several dozen miRNAs including miR-30 family, let-7 family, miR-21, miR-16, miR-155, miR-322 and Snord85 were changed during the differentiation process. These miRNAs were predicted to recognize osteogenic differentiation-, stemness-, epinegetics-, and cell cycle-related mRNAs, and were thus designated OstemiR. Among those OstemiR, the miR-30 family was classified into miR-30b/c and miR-30a/d/e groups on the basis of expression patterns during osteogenesis as well as mature miRNA structures. In silico prediction and subsequent qRT-PCR in stable miR-30d transfectants clarified that context-dependent targeting of miR-30d on known regulators of bone formation including osteopontin/spp1, lifr, ccn2/ctgf, ccn1/cyr61, runx2, sox9 as well as novel key factors including lin28a, hnrnpa3, hspa5/grp78, eed and pcgf5. In addition, knockdown of human OstemiR miR-541 increased Osteopontin/SPP1 expression and calcification in hMSC osteoblastic differentiation, indicating that miR-541 is a negative regulator of osteoblastic differentiation. These observations indicate stage-specific roles of OstemiR especially miR-541 and the miR-30 family on novel targets in osteogenesis.
    Full-text · Article · Mar 2013 · PLoS ONE
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    • "Then, nucleophosmin (NPM/B23) was re-discovered as being a specific RNA-binding regulator of the degradation of ccn2 mRNA. According to this study, down-regulation of NPM, which had been previously known only as a histone chaperon, eventually stabilizes the ccn2 mRNA to increase the steady-state level of the mRNA upon hypertrophic differentiation (Mukudai et al. 2008). This regulation is mediated by a cis-element in the 3′-UTR other than CAESAR and, interestingly, is chondrocyte specific. "
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    ABSTRACT: CCN2, a classical member of the CCN family of matricellular proteins, is a key molecule that conducts cartilage development in a harmonized manner through novel molecular actions. During vertebrate development, all cartilage is primarily formed by a process of mesenchymal condensation, while CCN2 is induced to promote this process. Afterwards, cartilage develops into several subtypes with different fates and missions, in which CCN2 plays its proper roles according to the corresponding microenvironments. The history of CCN2 in cartilage and bone began with its re-discovery in the growth cartilage in long bones, which determines the skeletal size through the process of endochondral ossification. CCN2 promotes physiological developmental processes not only in the growth cartilage but also in the other types of cartilages, i.e., Meckel's cartilage representing temporary cartilage without autocalcification, articular cartilage representing hyaline cartilage with physical stiffness, and auricular cartilage representing elastic cartilage. Together with its significant role in intramembranous ossification, CCN2 is regarded as a conductor of skeletogenesis. During cartilage development, the CCN2 gene is dynamically regulated to yield stage-specific production of CCN2 proteins at both transcriptional and post-transcriptional levels. New functional aspects of known biomolecules have been uncovered during the course of investigating these regulatory systems in chondrocytes. Since CCN2 promotes integrated regeneration as well as generation (=development) of these tissues, its utility in regenerative therapy targeting chondrocytes and osteoblasts is indicated, as has already been supported by experimental evidence obtained in vivo.
    Preview · Article · Feb 2011 · Journal of Cell Communication and Signaling
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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.
    Full-text · Article · Dec 2010 · Journal of Cellular Biochemistry
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