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Comparative Study
. 2008 Jul;58(7):2075-87.
doi: 10.1002/art.23504.

Differential expression of GADD45beta in normal and osteoarthritic cartilage: potential role in homeostasis of articular chondrocytes

Kosei Ijiri  1 , Luiz F Zerbini, Haibing Peng, Hasan H Otu, Kaneyuki Tsuchimochi, Miguel Otero, Cecilia Dragomir, Nicole Walsh, Benjamin E Bierbaum, David Mattingly, Geoff van Flandern, Setsuro Komiya, Thomas Aigner, Towia A Libermann, Mary B Goldring
Affiliations
Comparative Study

Differential expression of GADD45beta in normal and osteoarthritic cartilage: potential role in homeostasis of articular chondrocytes

Kosei Ijiri et al. Arthritis Rheum. 2008 Jul.

Abstract

Objective: Our previous study suggested that growth arrest and DNA damage-inducible protein 45beta (GADD45beta) prolonged the survival of hypertrophic chondrocytes in the developing mouse embryo. This study was undertaken, therefore, to investigate whether GADD45beta plays a role in adult articular cartilage.

Methods: Gene expression profiles of cartilage from patients with late-stage osteoarthritis (OA) were compared with those from patients with early OA and normal controls in 2 separate microarray analyses. Histologic features of cartilage were graded using the Mankin scale, and GADD45beta was localized by immunohistochemistry. Human chondrocytes were transduced with small interfering RNA (siRNA)-GADD45beta or GADD45beta-FLAG. GADD45beta and COL2A1 messenger RNA (mRNA) levels were analyzed by real-time reverse transcriptase-polymerase chain reaction, and promoter activities were analyzed by transient transfection. Cell death was detected by Hoechst 33342 staining of condensed chromatin.

Results: GADD45beta was expressed at higher levels in cartilage from normal donors and patients with early OA than in cartilage from patients with late-stage OA. All chondrocyte nuclei in normal cartilage immunostained for GADD45beta. In early OA cartilage, GADD45beta was distributed variably in chondrocyte clusters, in middle and deep zone cells, and in osteophytes. In contrast, COL2A1, other collagen genes, and factors associated with skeletal development were up-regulated in late OA, compared with early OA or normal cartilage. In overexpression and knockdown experiments, GADD45beta down-regulated COL2A1 mRNA and promoter activity. NF-kappaB overexpression increased GADD45beta promoter activity, and siRNA-GADD45beta decreased cell survival per se and enhanced tumor necrosis factor alpha-induced cell death in human articular chondrocytes.

Conclusion: These observations suggest that GADD45beta might play an important role in regulating chondrocyte homeostasis by modulating collagen gene expression and promoting cell survival in normal adult cartilage and in early OA.

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Figures

Figure 1
Figure 1
Comparison of gene expression patterns in cartilage samples from patients with early osteoarthritis (OA) and patients with late-stage OA. A, Growth arrest and DNA damage–inducible protein β (GADD45β) expression in cartilage samples from patients with early OA and patients with late-stage OA. RNA extracts from cartilage samples obtained from 3 patients with early OA (eOA1–eOA3) and 3 patients with late, or severe, OA (sOA1–sOA3) were analyzed by quantitative real-time polymerase chain reaction. Each sample was analyzed in triplicate. Bars show the mean and SD ratio of GADD45β to GAPDH. B, Self Organizing Map analysis of gene expression in cartilage samples from patients with early or severe OA. The algorithm generated 30 distinct clusters (c0–c29), each representing genes with a similar expression pattern. Each square in the grid corresponds to 1 cluster. The number in the top left of each square is the cluster ID. The yellow box around c0 marks the first cluster obtained in the analysis. The number in the top middle of each square is the number of genes in the cluster. Blue dots represent the average expression patterns of the genes in each cluster in (from left to right) severe OA sample 1, severe OA sample 2, severe OA sample 3, early OA sample 1, early OA sample 2, and early OA sample 3. Red lines represent the variation from the average expression pattern. Note that severe OA sample 3 is an outlier with respect to genes that are up-regulated in severe OA. C, Hierarchical clustering analysis of the microarray data from cartilage samples obtained from 3 patients with early OA and 3 patients with late OA, analyzed using Affymetrix GeneChip U133A. Up-regulated genes are shown in red; down-regulated genes are shown in green. Black indicates no change. GADD45β gene expression was up-regulated in early OA, along with genes involved in cell cycle regulation, such as DDX11, HRASLS3, and CCNG1, and in mitochondrial function, such as OXA1L and TOMM20, as well as other genes involved in intracellular functions. The genes encoding types I, II, III, and V collagen were all up-regulated in late OA compared with early OA, as were ASPN, MTN3, and the Wnt-associated genes, WIF1 and DKK3.
Figure 2
Figure 2
Microarray analysis of normal (N) and OA cartilage. RNA extracts from cartilage samples from 13 normal donors and 12 patients with late-stage OA were analyzed using Affymetrix GeneChip U133 Plus 2.0, and the data were subjected to bioinformatic analysis with dChip software, using a lower confidence bound (LCB) of >1.2. A, Fold change (FC) in expression of GADD45β, GADD45α, and GADD45γ transcripts in normal compared with OA cartilage. Values are the mean ± SEM. The probe ID is the Affymetrix identifier for separate probes on the microarray. B–D, Individual values in each normal and OA cartilage sample for each of the 3 GADD45β probes. See Figure 1 for other definitions.
Figure 3
Figure 3
Immunohistochemical analysis of GADD45β expression in representative specimens of human adult articular cartilage, including those used in the microarray analyses. A, Cartilage sections from a normal donor (Mankin grade 0) (a–c), a patient with early OA (Mankin grade 4) (d–f), and a patient with late-stage OA (Mankin grade 8) (g–i) were stained with toluidine blue or subjected to immunohistochemistry using anti-GADD45β or normal goat IgG (negative control). Arrows in b and e show single cells with nuclear staining (original magnification × 40). B, Sections with chondrocyte clusters in the middle zone from a patient with early OA (Mankin grade 4) (a–c) and an osteophyte from a patient with late OA (Mankin grade 8) (d–f) were stained with toluidine blue or immunostained with anti-GADD45β or normal goat IgG. Boxed areas in a and d are shown at higher magnification in b and e. See Figure 1 for other definitions.
Figure 4
Figure 4
Increased COL2A1 mRNA and promoter activity after small interfering RNA (siRNA) blockade of endogenous growth arrest and DNA damage–inducible protein β (GADD45β). A, Total RNA was extracted from cultures of C-28/I2 cells transduced with lentiviral siRNA–green fluorescent protein (GFP) (GFP–knockdown [KD]) or siRNA-GADD45β (GADD45β-KD) (left). Quantitative real-time reverse transcriptase–polymerase chain reaction analysis was performed to determine the levels of COL2A1 mRNA normalized to GAPDH mRNA. GFP-KD and GADD45β-KD C-28/I2 cells were transfected with pGL2-B/4.0, and COL2A1 promoter–driven luciferase activity was measured (right). B, C-28/I2 cells were cotransfected with pGL2-B/4.0 (COL2A1, spanning −577 to +3,428 bp) and pcDNA3-FLAG (control) or increasing amounts of GADD45β-FLAG, as indicated. Bars show the mean and SD of triplicate wells.
Figure 5
Figure 5
Induction of growth arrest and DNA damage–inducible protein β (GADD45β) by NF-κB in chondrocytes. C-28/I2 cells were cotransfected with pGL2-GADD45β promoter constructs −1,604 to +141 bp (A) and −496 to +141 bp (B), and the empty vector, pCI, or expression vector encoding the NF-κB subunit p65 alone, the NF-κB subunit p50 alone, or both p65 and p50. Bars show the mean and SD.
Figure 6
Figure 6
Growth arrest and DNA damage–inducible protein β (GADD45β) is a cell survival factor in human articular chondrocytes. Primary cultures were passaged at 2.5 × 104 cells/cm2, grown to confluence, transfected with small interfering RNA (siRNA)–GFP (siGFP) or siRNA-GADD45β oligos, and treated 72 hours later with tumor necrosis factor α (TNFα) for 24 hours. A, Cell death was evaluated by staining with Hoechst 33342 and counting the number of nuclei with brightly stained condensed chromatin. Bars show the mean ± SD percentage of cells showing nuclear condensation and fragmentation relative to the total number of cells counted (≥200 cells per data point). Results are from ≥4 independent experiments, each performed in triplicate. B, Representative photomicrographs of Hoechst-stained cells from 1 set of cultures isolated from a single patient are shown.
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