Posttranscriptional Regulation of Chicken ccn2 Gene Expression by Nucleophosmin/B23 during Chondrocyte Differentiation

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. CCN2 (connective tissue growth factor [CTGF]/hypertrophic chondrocyte-specific gene product 24 [Hcs24]) is a cysteine-rich secretory protein of 36 to 38 kDa that has four distinct modules, i.e., insulin-like growth factor-binding protein-likes, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and C-terminal modules (4,6,54,68,69). CCN2 is a member of the CCN family (reviewed in references 6, 38, 53, 54, 68, and 69), which comprises ccn1 (cef-10/cyr61 [37,66]), ccn2 (ctgf/hcs24/fisp12 [5,46,57]), ccn3 (nov [25]), ccn4 (elm-1/wisp-1 [20,52]), ccn5 (ctgf-l/wisp-2/cop1 [52,75]), and ccn6 (wisp-3 [52]). CCN2 was initially isolated from angioendothelial cells as a growth factor related to plateletderived growth factor (PDGF) and was revealed to have PDGF-like mitogenic and chemotactic activities toward fibroblasts (17,23,28,33,54). However, recent studies have revealed that CCN2 is a multifunctional factor that regulates the growth and/or differentiation, chemotaxis, adhesion of various cells, and extracellular matrix formation by various cells, including vascular endothelial cells (2,29,54,64). Furthermore, we showed earlier that CCN2 plays an important role in the growth and differentiation of chondrocytes and osteoblasts during endochondral ossification (46-48).As to CCN2, it has been suggested that its gene expression is regulated at multiple steps, such as transcriptional, posttranscriptional, and translational stages (1), for playing its multiple roles mentioned above. For instance, transforming growth factor ␤ (TGF-␤) induces the expression of CCN2 (28,46), and a few TGF-␤ response elements have been found in the ccn2 promote...

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“…In fact, it has been shown that NPM is selectively deposited on mRNA during polyadenylation, but the biological significance of this binding remains unknown (Palaniswamy et al, 2006). More recently, NPM has been reported to bind the 3 0 untranslated region of the product of the chicken connective tissue growth factor gene, ccn2, triggering its degradation, and this mechanism contributes to modulate chondrocyte differentiation (Mukudai et al, 2008).…”

CCN2 in orofacial tissue development and remodeling

“…MMP-3, an ECMdegrading enzyme, was also found as a novel target to regulate CCN2 gene expression; for this enzyme is taken up by cells and directly binds to and controls the transcription of the CCN2 gene [69]. Also at post-transcriptional stages, several proteins and noncoding RNAs are known to repress CCN2 gene expression, either by accelerating the degradation or repressing the translation of the CCN2 mRNA [70,71]. Nucleophosmin/B23, a histone chaperone shuttling between the nucleus and cytoplasm, was unexpectedly found to interact with and degrade CCN2 mRNA in chicken chondrocytes [71].…”

“…Also at post-transcriptional stages, several proteins and noncoding RNAs are known to repress CCN2 gene expression, either by accelerating the degradation or repressing the translation of the CCN2 mRNA [70,71]. Nucleophosmin/B23, a histone chaperone shuttling between the nucleus and cytoplasm, was unexpectedly found to interact with and degrade CCN2 mRNA in chicken chondrocytes [71]. Several miRNAs have also been identified as post-transcriptional repressors of the CCN2 gene [72,73].…”

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“…18 These studies suggested a functional interplay between the central ribonuclease domain and the C-terminal domain, with the latter seemingly implicated in substrate recognition and positioning. More recently, NPM1 was also found to be associated to mRNA during polyadenylation 40 and shown to bind the 3 0 -UTR region of the chicken connective tissue growth factor gene product promoting its degradation, 41 suggesting that the NPM1 RNA-binding activity is not confined to pre-rRNA processing and ribosome assembly.…”