Paraspeckle protein p54nrb links Sox9-mediated transcription with RNA processing during chondrogenesis in mice
Kenji Hata, … , Vincent R. Harley, Toshiyuki Yoneda
Kenji Hata, … , Vincent R. Harley, Toshiyuki Yoneda
Published August 1, 2008
Citation Information: J Clin Invest. 2008;118(9):3098-3108. https://doi.org/10.1172/JCI31373.
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Research Article Bone biology

Paraspeckle protein p54nrb links Sox9-mediated transcription with RNA processing during chondrogenesis in mice

Abstract

The Sox9 transcription factor plays an essential role in promoting chondrogenesis and regulating expression of chondrocyte extracellular-matrix genes. To identify genes that interact with Sox9 in promoting chondrocyte differentiation, we screened a cDNA library generated from the murine chondrogenic ATDC5 cell line to identify activators of the collagen, type II, α 1 (Col2a1) promoter. Here we have shown that paraspeckle regulatory protein 54-kDa nuclear RNA-binding protein (p54nrb) is an essential link between Sox9-regulated transcription and maturation of Sox9-target gene mRNA. We found that p54nrb physically interacted with Sox9 and enhanced Sox9-dependent transcriptional activation of the Col2a1 promoter. In ATDC5 cells, p54nrb colocalized with Sox9 protein in nuclear paraspeckle bodies, and knockdown of p54nrb suppressed Sox9-dependent Col2a1 expression and promoter activity. We generated a p54nrb mutant construct lacking RNA recognition motifs, and overexpression of mutant p54nrb in ATDC5 cells markedly altered the appearance of paraspeckle bodies and inhibited the maturation of Col2a1 mRNA. The mutant p54nrb inhibited chondrocyte differentiation of mesenchymal cells and mouse metatarsal explants. Furthermore, transgenic mice expressing the mutant p54nrb in the chondrocyte lineage exhibited dwarfism associated with impairment of chondrogenesis. These data suggest that p54nrb plays an important role in the regulation of Sox9 function and the formation of paraspeckle bodies during chondrogenesis.

Authors

Kenji Hata, Riko Nishimura, Shuji Muramatsu, Akio Matsuda, Takuma Matsubara, Katsuhiko Amano, Fumiyo Ikeda, Vincent R. Harley, Toshiyuki Yoneda

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Figure 6

Role of p54nrb in the regulation of chondrocyte differentiation.

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Role of p54nrb in the regulation of chondrocyte differentiation. (A)...
(A) The p54nrb-induced Col2a1 and Acan in ATDC5 cells. ATDC5 cells were transfected as indicated, and then subjected to RT-PCR analyses. (B) The p54nrb-induced type IIA and IIB forms of Col2a1 in ATDC5 cells. ATDC5 cells were transfected as indicated, and then subjected to RT-PCR analyses using the specific primers (see Methods) for Col2a1 (upper panel; the primer set can distinguish 2 isoforms of Col2a1), Col2a1 (middle panel; the primer set cannot distinguish 2 isoforms of Col2a1), or β-actin. (C and D) ATDC5 cells were infected with Sox9 and/or Myc-tagged wild-type or a Myc-tagged mutant p54nrb (Δ224) adenovirus and then subjected to northern (C) or western (D) blotting, respectively. (E) Knockdown of p54nrb suppressed Sox9-induced Col2a1 expression. ATDC5 cells were transfected with expression vectors as indicated, and then total RNA of the cells was determined by RT-PCR analyses. (F) The p54nrb mutant blocked chondrocyte differentiation. C3H10T1/2 cells were infected with Myc-tagged wild-type or mutant p54nrb (Δ224) adenovirus in the presence or absence of BMP2. The cells were examined by alcian blue staining. (G) No effects of p54nrb on osteoblastogenesis. C2C12 cells infected with Myc-tagged wild-type or mutant p54nrb (Δ224) adenovirus in the presence or absence of BMP2. The cells were examined by alkaline phosphatase staining. (H and I) Requirement of p54nrb for chondrogenesis. Metatarsals isolated from mouse embryo were infected with control, Myc-tagged wild-type p54nrb, or a Flag-tagged mutant p54nrb (Δ224) adenovirus, incubated for 6 days, and subjected to immunostaining (H), histological analysis (I), and statistically analyses of the samples (J). PZ, proliferating zone; HZ, hypertrophic zone. All data were analyzed by ANOVA, followed by Student’s t test. Values shown are mean ± SD. (*P < 0.05, **P < 0.01 versus control). Original magnification, ×50 (G); ×40 (I).