Differential Expression of GADD45\u3ci\u3eβ\u3c/i\u3e 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 45β (GADD45β) prolonged the survival of hypertrophic chondrocytes in the developing mouse embryo. This study was undertaken, therefore, to investigate whether GADD45β 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 GADD45β was localized by immunohistochemistry. Human chondrocytes were transduced with small interfering RNA (siRNA)–GADD45β or GADD45β-FLAG. GADD45β 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—GADD45β 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 GADD45β. In early OA cartilage, GADD45β 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, GADD45β down-regulated COL2A1 mRNA and promoter activity. NF-κB overexpression increased GADD45β promoter activity, and siRNA-GADD45β decreased cell survival per se and enhanced tumor necrosis factor α–induced cell death in human articular chondrocytes. Conclusion—These observations suggest that GADD45β 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

Differential Expression of GADD45\u3ci\u3eβ\u3c/i\u3e 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 45β (GADD45β) prolonged the survival of hypertrophic chondrocytes in the developing mouse embryo. This study was undertaken, therefore, to investigate whether GADD45β 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 GADD45β was localized by immunohistochemistry. Human chondrocytes were transduced with small interfering RNA (siRNA)–GADD45β or GADD45β-FLAG. GADD45β 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—GADD45β 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 GADD45β. In early OA cartilage, GADD45β 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, GADD45β down-regulated COL2A1 mRNA and promoter activity. NF-κB overexpression increased GADD45β promoter activity, and siRNA-GADD45β decreased cell survival per se and enhanced tumor necrosis factor α–induced cell death in human articular chondrocytes. Conclusion—These observations suggest that GADD45β 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

CCAAT/enhancer binding protein β (C/EBPβ) regulates the transcription of growth arrest and DNA damage-inducible protein 45 β (GADD45β) in articular chondrocytes

AbstractOsteoarthritis (OA) is a whole joint disease characterized by cartilage degradation, which causes pain and disability in older adults. Our previous work showed that growth arrest and DNA damage-inducible protein 45 β (GADD45β) is upregulated in chondrocyte clusters in OA cartilage, especially in the early stage of this disease. CCAAT/enhancer binding protein β (C/EBPβ) is expressed in the hypertrophic growth plate chondrocytes and functions in synergy with GADD45β. Here, the presence and localization of these proteins was assessed by immunohistochemistry using articular cartilage from OA patients, revealing colocalization of C/EBPβ and GADD45β in OA chondrocytes. GADD45β promoter analysis was performed to determine whether C/EBPβ directly regulates GADD45β transcription. Furthermore, we analyzed the effect of C/EBPβ on Gadd45β gene regulation in articular chondrocytes in vivo and in vitro. Immunohistochemical analysis of C/ebpβ-haploinsufficient mice (C/ebpβ+/−) cartilage showed that C/ebpβ haploinsufficiency led to reduced Gadd45β gene expression in these cells. In vitro, we evaluated the effects of conditional C/EBPβ overexpression driven by the cartilage oligomeric matrix protein (Comp) promoter in mComp-tTA;pTRE-Tight-BI-DsRed-mC/ebpβ transgenic mice. C/EBPβ overexpression significantly stimulated Gadd45β gene expression in articular chondrocytes. Taken together, our data demonstrate that C/EBPβ plays a central role in controlling Gadd45β gene expression in these cells

Identification of a Crucial Site for Synoviolin Expression

Synoviolin is an E3 ubiquitin ligase localized in the endoplasmic reticulum (ER) and serving as ER-associated degradation system. Analysis of transgenic mice suggested that synoviolin gene dosage is implicated in the pathogenesis of arthropathy. Complete deficiency of synoviolin is fatal embryonically. Thus, alternation of Synoviolin could cause breakdown of ER homeostasis and consequently lead to disturbance of cellular homeostasis. Hence, the expression level of Synoviolin appears to be important for its biological role in cellular homeostasis under physiological and pathological conditions. To examine the control of protein level, we performed promoter analysis to determine transcriptional regulation. Here we characterize the role of synoviolin transcription in cellular homeostasis. The Ets binding site (EBS), termed EBS-1, from position −76 to −69 of the proximal promoter, is responsible for synoviolin expression in vivo and in vitro. Interestingly, transfer of EBS-1 decoy into NIH 3T3 cells conferred not only the repression of synoviolin gene expression but also a decrease in cell number. Fluorescence-activated cell sorter analysis using annexin V staining confirmed the induction of apoptosis by EBS-1 decoy and demonstrated recovery of apoptosis by overexpression of Synoviolin. Our results suggest that transcriptional regulation of synoviolin via EBS-1 plays an important role in cellular homeostasis. Our study provides novel insight into the transcriptional regulation for cellular homeostasis

Synoviolin/Hrd1, an E3 ubiquitin ligase, as a novel pathogenic factor for arthropathy

Rheumatoid arthritis (RA) is one of the most critical articular diseases with synovial hyperplasia followed by impairment of quality of life. However, the mechanism(s) that regulates synovial cell outgrowth is not fully understood. To clarify its mechanism(s), we carried out immunoscreening by using antirheumatoid synovial cell antibody and identified and cloned “Synoviolin/Hrd1”, an E3 ubiquitin ligase. Synoviolin/Hrd1 was highly expressed in the rheumatoid synovium, and mice overexpressing this enzyme developed spontaneous arthropathy. Conversely, synoviolin/hrd1(+/-) mice were resistant to collagen-induced arthritis by enhanced apoptosis of synovial cells. We conclude that Synoviolin/Hrd1 is a novel causative factor for arthropathy by triggering synovial cell outgrowth through its antiapoptotic effects. Our findings provide a new pathogenetic model of RA and suggest that Synoviolin/Hrd1 could be targeted as a therapeutic strategy for RA

A cis-regulatory site downregulates PTHLH in translocation t(8;12)(q13;p11.2) and leads to Brachydactyly Type E

Parathyroid hormone-like hormone (PTHLH) is an important chondrogenic regulator; however, the gene has not been directly linked to human disease. We studied a family with autosomal-dominant Brachydactyly Type E (BDE) and identified a t(8;12)(q13;p11.2) translocation with breakpoints (BPs) upstream of PTHLH on chromosome 12p11.2 and a disrupted KCNB2 on 8q13. We sequenced the BPs and identified a highly conserved Activator protein 1 (AP-1) motif on 12p11.2, together with a C-ets-1 motif translocated from 8q13. AP-1 and C-ets-1 bound in vitro and in vivo at the derivative chromosome 8 breakpoint [der(8) BP], but were differently enriched between the wild-type and BP allele. We differentiated fibroblasts from BDE patients into chondrogenic cells and found that PTHLH and its targets, ADAMTS-7 and ADAMTS-12 were downregulated along with impaired chondrogenic differentiation. We next used human and murine chondrocytes and observed that the AP-1 motif stimulated, whereas der(8) BP or C-ets-1 decreased, PTHLH promoter activity. These results are the first to identify a cis-directed PTHLH downregulation as primary cause of human chondrodysplasia