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


CONCLUSIONS
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.
Ijiri
Kosei
K
Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
Zerbini
Luiz F
LF
Peng
Haibing
H
Otu
Hasan H
HH
Tsuchimochi
Kaneyuki
K
Otero
Miguel
M
Dragomir
Cecilia
C
Walsh
Nicole
N
Bierbaum
Benjamin E
BE
Mattingly
David
D
van Flandern
Geoff
G
Komiya
Setsuro
S
Aigner
Thomas
T
Libermann
Towia A
TA
Goldring
Mary B
MB
D000368:Aged; D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis* / Q000235:genetics; D002358:Cartilage, Articular / Q000378:metabolism* / Q000473:pathology; D019902:Chondrocytes / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling; D006706:Homeostasis; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D010003:Osteoarthritis / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012720:Severity of Illness Index; D000097642:GADD45 Proteins
D000943:Antigens, Differentiation; C086470:GADD45B protein, human; D000097642:GADD45 Proteins