Paraspeckle protein p54nrb links Sox9-mediated transcription with RNA processing during chondrogenesis in mice - PubMed (original) (raw)

Kenji Hata et al. J Clin Invest. 2008 Sep.

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, alpha 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 p54(nrb) 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.

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Figures

Figure 2

Figure 2. Importance of association of p54nrb with Sox9 in upregulation of Col2a1 gene promoter activity.

(A) Inhibition of p54nrb-stimulated Col2a1 promoter activity by dominant-negative Sox9 (DN). Luciferase activity of ATDC5 cell lysates transfected with Col2a1 luciferase construct, together with expression vectors as indicated was measured. (B) No association of dominant-negative Sox9 with p54nrb. The cell lysates expressing wild-type or the mutants of HA-Sox9 were precipitated (Ppt) with His-tag-p54nrb protein, and then the precipitates were determined by immunoblotting with anti-HA antibody. ΔHMG, a mutant lacking the HMG domain. (C) Schematic diagram of the mutants of p54nrb. ΔM, Δ224, and Δ313 are mutants of p54nrb. (D) Analysis of binding domain of p54nrb with Sox9. The cell lysates expressing wild-type or mutants of p54nrb were precipitated with tandem affinity purification–tagged (TAP-tagged) Sox9 protein, and then the precipitates were determined by immunoblotting with anti-Myc antibody. (E) The mutant p54nrb (ΔM) failed to transactivate the transcriptional activity of Sox9. ATDC5 cells were transfected with constructs as indicated, and luciferase activity of cell lysates was measured. (F) Knockdown of p54nrb by shRNA. shRNA expression vector for GFP or p54nrb (shGFP or shp54nrb) was transfected into ATDC5 cells, and the total RNA of the cells was determined by RT-PCR analyses. (G) Knockdown of p54nrb by shRNA. ATDC5 cells were transfected with shGFP or shp54nrb, and the cell lysates were examined by immunoblotting with anti-p54nrb and β-actin antibodies. (H) Inhibition of Sox9-dependent Col2a1 promoter activity by knockdown of p54nrb. Luciferase activity was measured in cell lysates transfected with expression vectors as indicated.

Figure 3

Figure 3. Colocalization of p54nrb with Sox9 in paraspeckle nuclear bodies.

(A) Colocalization of p54nrb with PSP1 in the nucleus of ATDC5 cells. ATDC5 cells transfected with Venus-tagged p54nrb (Venus-p54nrb) and DsRed-tagged PSP1 (DsRed-PSP1) were monitored under a fluorescence microscope. (B) p54nrb is not localized with SC-35. ATDC5 cells transfected with Venus-tagged p54nrb were immunostained with anti–SC-35 antibody. (C) Colocalization of p54nrb and Sox9 in the nucleus. ATDC5 cells transfected with Venus-tagged p54nrb and DsRed-tagged Sox9 (DsRed-Sox9) were monitored under a fluorescence microscope. (D and E) Colocalization of p54nrb and Sox9 in the nucleus. C3H10T1/2 cells transfected with Venus-tagged p54nrb and DsRed-tagged Sox9 were monitored under a confocal microscope. C3H10T1/2 cells expressing both Venus-p54nrb and DsRed-Sox9 show smaller size of speckle than C3H10T1/2 cells expressing only Venus-p54nrb (D). Nucleuses of cells were stained with DAPI. (F) Colocalization of p54nrb and Sox9 in murine primary chondrocytes. Primary chondrocytes isolated from mouse ribs were immunostained with anti-p54nrb and anti-Sox9 antibodies and photographed under a confocal microscope. (G) Colocalization of Sox9 with PSP1 in the nucleus of ATDC5 cells. ATDC5 cells transfected with DsRed-tagged Sox9 and Venus-tagged PSP1 (Venus-PSP1) were monitored under a fluorescence microscope. (H) Sox9 does not affect localization of SC-35. ATDC5 cells transfected with DsRed-tagged Sox9 were immunostained with anti-SC-35 antibody. Original magnification, ×400.

Figure 1

Figure 1. Physical and functional interactions of p54nrb with Sox9.

(A) Stimulation of Col2a1 promoter activity by cDNA clones isolated from ATDC5. ATDC5 cells were transfected with each cDNA together with the _Col2a1_-luciferase construct, and luciferase activity of cell lysates was measured. Clone 3 (cl 3) is p54nrb. Cont, control. (B) Costimulatory effect of p54nrb and Sox9 on Col2a1 promoter activity. ATDC5 cells were transfected with p54nrb together with Sox9, and luciferase activity of cell lysates was measured. (C) No upregulation of Col2a1 promoter activity by p54nrb in HeLa cells. HeLa cells were transfected with p54nrb together with Sox9, and luciferase activity of cell lysates was measured. (D) Absence of effect of p54nrb on transcriptional activity of Runx2. The osteocalcin gene promoter luciferase constructs were transfected into C3H10T1/2 cells with the plasmid as indicated. The luciferase activity of cell lysates was measured. (E) Physical association of p54nrb with Sox9. Cell lysates expressing Myc-p54nrb, HA-Sox9, or both were determined by immunoblotting with anti-HA antibody, followed by immunoprecipitation with anti-Myc antibody.

Figure 4

Figure 4. Processing of Col2a1 mRNA by p54nrb.

(A) Col2a1 mRNA processing by p54nrb. A minigene construct of the Col2a1 gene was transfected with expression plasmids as indicated into ATDC5 cells. Total RNA isolated from the cells was determined by RT-PCR analyses using the primers (see Methods) specific for the minigene products (upper panel) or β-actin (bottom panel) Ex1, exon 1. (B) Effect of wild-type and a mutant p54nrb on processing of Col2a1 mRNA. A minigene construct of the Col2a1 gene was transfected with the expression vectors as indicated into ATDC5 cells. Expression of spliced minigene product (upper panel) and nonspliced minigene product (bottom panel) was determined by real-time PCR using the primers (see Methods) specific for the spliced and nonspliced minigene products. Expression levels were normalized with β-actin expression. (C) Effect of p54nrb on processing of fibronectin mRNA. HEK293 cells were transfected with fibronectin minigene construct (7iBi) together with mock (Cont), SRP40 expression vector, or p54nrb expression vector. Total RNA isolated from the cells was determined by RT-PCR analyses using the primers (see Methods) specific for the minigene products (upper panel) or GAPDH (bottom panel).

Figure 5

Figure 5. Impairment of paraspeckle formation and Col2a1 mRNA processing by a mutant p54nrb (Δ244).

(A) Impairment of paraspeckle body formation by a p54nrb mutant. ATDC5 cells transfected with Venus-tagged wild-type or mutant p54nrb and DsRed-PSP1 were visualized under a fluorescence microscope. Nucleuses of cells were stained with DAPI. (B) Effect of the mutant p54nrb on proliferation of ATDC5 cells. Control, wild-type p54nrb, or the mutant p54nrb were transfected into ATDC5 cells, and the growth of the cells was determined by cell proliferation assay. (C) Wild-type and a mutant p54nrb does not affect localization of SC-35. ATDC5 cells transfected with Venus-tagged wild-type or mutant p54nrb were immunostained with anti–SC-35 antibody. (D) Upregulation of transcriptional activity of Sox9 by a p54nrb mutant. ATDC5 cells were transfected with wild-type and a mutant p54nrb together with Sox9, and luciferase activity of cell lysates was measured. (E) Colocalization of a mutant p54nrb with Sox9. ATDC5 cells transfected with DsRed-tagged Sox9 and Venus-tagged p54nrb mutant were monitored under a fluorescence microscope. (F) Impairment of Col2a1 mRNA processing by a mutant p54nrb. A minigene construct of the Col2a1 gene was transfected with expression plasmids as indicated into ATDC5 cells. Total RNA isolated from the cells was determined by RT-PCR analyses using the primers (see Methods) specific for the minigene products (upper panel) or β-actin (bottom panel). Original magnification, ×400 (A, C, and E).

Figure 6

Figure 6. Role of p54nrb in the regulation of chondrocyte differentiation.

(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).

Figure 7

Figure 7. Impaired chondrogenesis in the transgenic mice expressing the p54nrb mutant.

(A) The expression of the transgene in the chondrocytes isolated from the transgenic mice. Total RNA of chondrocytes isolated from wild-type or the transgenic mice was determined by RT-PCR analysis using specific primer (see Methods) for the transgene (Δ224) (upper panel) or β-actin (bottom panel). (B) Specific expression of p54nrb mutant in chondrocytes of the transgenic mice. Total RNA isolated from brain (lane 1), kidney (lane 2), heart (lane 3), liver (lane 4), spleen (lane 5), chondrocytes (lane 6), or osteoblasts (lane 7) of the transgenic mice was determined by RT-PCR using specific primer (see Methods) for transgene (Δ224) (upper panel) or β-actin (bottom panel). (C) Overexpression of the mutant p54nrb in the transgenic mice. Total RNA of chondrocytes isolated from wild-type or the transgenic mice was determined by real-time PCR analysis using a Taqman probe, which recognizes both intact and the mutant p54nrb. (D and E) Dwarfism of the transgenic mice at birth. (F and G) Dwarfism of the transgenic mice at 3 weeks. D and F are macroscopic photographs and E and G are x-rays of the mice. (H) Body size of the transgenic mice. (I) H&E-stained section of the tibias of wild-type and transgenic mice at 2 weeks. (J) Col2 expression in the tibias of wild-type and transgenic mice. (K) Reduction in Col2α1 expression in the transgenic mice. Total RNA of chondrocytes isolated from wild-type or the transgenic mice was determined by real-time PCR analysis using a Taqman probe for Col2a1. Original magnification, ×40 (I and J).

Figure 8

Figure 8. Delayed enchondral ossification in the transgenic mice.

(A) The sections of wild-type and the transgenic mice at neonatal period were subjected to H&E, alcian blue, or von Kossa staining. (B) The sections of wild-type and the transgenic mice at E15 were determined by in situ hybridization (Col2a1, Col10a1, PTHR1, Ihh), H&E staining, and von Kossa staining. (C) Normal proliferation of chondrocytes in wild-type and the transgenic mice at E15. The sections of wild-type and the transgenic mice at E15 were determined by immunostaining with anti-PCNA antibody. Original magnification, ×25 (A); ×40 (B); ×20 (C, left panel of WT and Tg); ×40 (C, right panel of WT and Tg).

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