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. 2005 Nov 18;280(46):38544-55.
doi: 10.1074/jbc.M504202200. Epub 2005 Sep 2.

A novel role for GADD45beta as a mediator of MMP-13 gene expression during chondrocyte terminal differentiation

Kosei Ijiri  1 , Luiz F Zerbini, Haibing Peng, Ricardo G Correa, Binfeng Lu, Nicole Walsh, Yani Zhao, Noboru Taniguchi, Xu-Ling Huang, Hasan Otu, Hong Wang, Jian Fei Wang, Setsuro Komiya, Patricia Ducy, Mahboob U Rahman, Richard A Flavell, Ellen M Gravallese, Peter Oettgen, Towia A Libermann, Mary B Goldring
Affiliations

A novel role for GADD45beta as a mediator of MMP-13 gene expression during chondrocyte terminal differentiation

Kosei Ijiri et al. J Biol Chem. .

Abstract

The growth arrest and DNA damage-inducible 45beta (GADD45beta) gene product has been implicated in the stress response, cell cycle arrest, and apoptosis. Here we demonstrated the unexpected expression of GADD45beta in the embryonic growth plate and uncovered its novel role as an essential mediator of matrix metalloproteinase-13 (MMP-13) expression during terminal chondrocyte differentiation. We identified GADD45beta as a prominent early response gene induced by bone morphogenetic protein-2 (BMP-2) through a Smad1/Runx2-dependent pathway. Because this pathway is involved in skeletal development, we examined mouse embryonic growth plates, and we observed expression of Gadd45beta mRNA coincident with Runx2 protein in pre-hypertrophic chondrocytes, whereas GADD45beta protein was localized prominently in the nucleus in late stage hypertrophic chondrocytes where Mmp-13 mRNA was expressed. In Gadd45beta(-/-) mouse embryos, defective mineralization and decreased bone growth accompanied deficient Mmp-13 and Col10a1 gene expression in the hypertrophic zone. Transduction of small interfering RNA-GADD45beta in epiphyseal chondrocytes in vitro blocked terminal differentiation and the associated expression of Mmp-13 and Col10a1 mRNA in vitro. Finally, GADD45beta stimulated MMP-13 promoter activity in chondrocytes through the JNK-mediated phosphorylation of JunD, partnered with Fra2, in synergy with Runx2. These observations indicated that GADD45beta plays an essential role during chondrocyte terminal differentiation.

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Figures

FIGURE 1
FIGURE 1. Specific up-regulation of GADD45β mRNA by BMP-2 in chondrocytes
Monolayer cultures of the human chondrocyte cell line C-28/I2 were treated as indicated, and total RNA extracts were analyzed by real time PCR. Each value was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the same sample and shown as the mean ± S.D. of triplicate cultures. A, GADD45β mRNA levels were increased in cultures of the human chondrocyte cell line, C-28/I2, after treatment with BMP-2 for 1 h but not with EGF, bFGF, or IGF-I. B and C, incubation of C-28/I2 cells with BMP-2 for 1 h did not increase the levels of GADD45α or GADD45γ mRNA. D, time course of BMP-2-induced GADD45β mRNA in C-28/I2 cells showed transient induction with a peak at 1 h. E, treatment of primary human articular chondrocytes with BMP-2 for 1 h increased the levels of GADD45β mRNA by 2-fold.
FIGURE 2
FIGURE 2. The induction of GADD45β promoter activity by BMP-2 requires signaling by Smad1 interacting with Runx2
A, the pGL2-GADD45β promoter (−1604/+141 bp) was cotransfected with expression vectors encoding the BMP type IB receptor (alk6), Smad1, or Smad6 alone or in combination, or Runx2 or ΔRunx2 alone or in combination. The results show the means ± S.D. of determinations of samples from three or more separate wells. B, the C-28/I2 cells were incubated without (−) or with (+) BMP-2 for 1 h, and total lysates were analyzed on Western blots (WB) using antibodies against phospho-Smad1/5/8 and total Smad1. C, total cell lysates were subjected to immunoprecipitation (IP) with anti-Runx2 and analyzed on Western blots using anti-phosphoserine or Smad1 antibody.
FIGURE 3
FIGURE 3. Expression of Gadd45β mRNA together with Runx2 protein in prehypertrophic chondrocytes and intracellular GADD45β protein in the hypertrophic chondrocytes that express Mmp-13 mRNA
Adjacent longitudinal sections from mouse embryonic femurs or tibiae (15.5 dpc) were analyzed by immunohistochemistry and in situ hybridization to illustrate the expression of GADD45β in relation to Runx2 protein and MMP-13 mRNA in proliferating (pro), prehypertrophic (pre-hy), and hypertrophic (hyp) zones indicated in A. Antisense Gadd45β (A and B) and anti-GADD45β antibody (D) were used to detect Gadd45β mRNA, and protein and anti-Runx2 antibody (C) detected Runx2 protein in mouse femurs. Sense Gadd45β (insets, A and B), normal goat IgG (inset, C), and normal rabbit IgG (inset, D) were applied as negative controls. Tibiae at 15.5 dpc were stained with toluidine blue (T.Blue, E). GADD45β protein was detected in late stage hypertrophic chondrocytes (F and J), in which Mmp-13 mRNA was also detected using antisense MMP-13 (H and K). Normal goat IgG (G) and sense Mmp-13 (I) were applied as negative controls. Brown staining indicates immunodetectable protein, and purple staining indicates a positive signal using antisense probe. Original magnification: A, E–I, ×40; B–D, F, and G, ×200.
FIGURE 4
FIGURE 4. Defective mineralization accompanies decreased Mmp-13 mRNA expression in hypertrophic chondrocytes of Gadd45β−/− mouse embryos at 15 dpc
Tibiae from WT and Gadd45β−/− mouse embryos at 15.5 dpc were examined by toluidine blue staining (A and D), von Kossa staining (B and E), and in situ hybridization for Mmp-13 mRNA (C, F, G, and H). Original magnifications: A–F, ×40; G and H, ×200.
FIGURE 5
FIGURE 5. Gadd45β−/− mouse embryos at 16. 5 dpc exhibit decreased bone growth associated with decreased levels of Mmp-13 and Col10a1 mRNA in hypertrophic chondrocytes
Whole mounted embryos at 16.5 dpc were stained with Alcian Blue and Alizarin Red to show decreased cartilage matrix and mineralization in ribs (A, arrowheads), forearms (B), and hind limbs (C) of Gadd45β−/− compared with WT mice. Adjacent longitudinal sections from the tibia at 16.5 dpc showed a smaller hypertrophic zone by toluidine blue (T.Blue) staining (D) accompanied by markedly decreased levels of Mmp-13 mRNA (E, arrowhead) and Col10a1 mRNA in Gadd45β−/− mouse embryos compared with WT. Original magnifications: D, ×40; E, ×200.
FIGURE 6
FIGURE 6. GADD45β is required for hypertrophic chondrocyte differentiation in vitro and associated expression of Mmp-13 and Col10a1 mRNA
A, murine rib growth plate chondrocytes were infected with lentivirus encoding siRNA-GFP to generate GFP KD cells or with siRNA-GADD45β to generate GADD45β KD cells and cultured as three-dimensional pellets for 3, 14, and 21 days. Toluidine blue staining showed the progressive hypertrophy in cultures of GFP KD chondrocytes but not in the cultures of GADD45β KD chondrocytes. Magnification is ×40. B, after 21 days of culture, toluidine blue (T.Blue) staining and the levels of Col10a1 and Mmp-13 mRNA, detected by in situ hybridization using antisense probes, were markedly decreased in GADD45β KD compared with GFP KD cells. Magnification is ×200.
FIGURE 7
FIGURE 7. GADD45β positively regulates AP-1 activity and JunD/Fra2 binding in chondrocytes
The AP-1-driven luciferase reporter vector (pAP-1-Luc) was cotransfected in C-28/I2 cells with the GADD45β expression vector (GADD45β-FLAG) at 50 and 100 ng/ml (A) or with GADD45β-FLAG alone or together with siRNA-GADD45β (B). C, extracts were prepared from uninfected C-28/I2 cells (1st and 2nd lanes), cells infected with lentiviral siRNA-GFP (GFP KD; 3rd and 5th lanes), or siRNA-GADD45β (GADD45β KD; 4th and 6th lanes). Cells were then transfected with GFP-FLAG (control) or GADD45β-FLAG, as indicated. AP-1 binding activity was examined by EMSA using the double-stranded AP-1 consensus oligonucleotide as labeled probe. The cells extracted for the 5th and 6th lanes (lower exposure of gel shift using probe labeled at different time) were not transfected to show that decreasing endogenous GADD45β also decreases AP-1 binding activity. D, supershift analysis of binding activities in GADD45β-expressing C-28/I2 cells was performed using antibodies against different AP-1 family members. Note that the Fra1, JunB, and JunD antibodies produce supershifts, whereas the Fra2 antibody produces a block shift. E, the C-28/I2 cells were cotransfected with pAP-1-Luc together with expression vectors encoding Fra1, Fra2, JunD, and JunB alone or in combination in the absence or presence of the GADD45β-FLAG. Luciferase activities are shown as means ± S.D. of replicates from representative transfections. *, p < 0.05; **, p < 0.01; by analysis of variance with subsequent Dunnett test in multiple comparisons. Comparisons in E are with respective controls containing single expression vectors.
FIGURE 8
FIGURE 8. JNK activity is necessary for JunD activation by GADD45β
A, extracts from C-28/I2 cells expressing GADD45β-FLAG or siRNA-GADD45β were analyzed by Western blotting using antibodies against phosphorylated (phospho) or total JunD. GADD45β-expressing cells were also treated with inhibitors of ERK (PD98059), p38 (SB203580), and JNK (SP600125) to examine the roles of the different kinases in JunD phosphorylation by GADD45β. B, kinase activities were analyzed on Western blots to show increased JNK (phospho-c-Jun) and ERK (phospho-Elk) activities in GADD45β-FLAG cells and decreased activities in cells expressing siRNA-GADD45β. Note that p38 activity (phospho-ATF-2) did not change.
FIGURE 9
FIGURE 9. JunD-dependent MMP-13 promoter activation by GADD45β in cooperation with Runx2 in the ATDC5 chondrogenic cell line
The ATDC5 cells were cotransfected with the pGL2-MMP-13 promoter (−1007 bp/+26 bp) construct and expression vectors encoding GADD45β and Runx2 (A), siRNA-GADD45β (B), Runx2 and JunD (C), or GADD45β and dominant negative (DN) JunD (D). The results show the means ± S.D. of determinations in triplicate wells from representative experiments. Empty expression vector controls or promoter-less pGL2-B did not induce or express any significant luciferase activity (not shown).
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