RNF168 cooperates with RNF8 to mediate FOXM1 ubiquitination and degradation in breast cancer epirubicin treatment.

The forkhead box M1 (FOXM1) transcription factor has a central role in genotoxic agent response in breast cancer. FOXM1 is regulated at the post-translational level upon DNA damage, but the key mechanism involved remained enigmatic. RNF168 is a ubiquitination E3-ligase involved in DNA damage response. Western blot and gene promoter-reporter analyses showed that the expression level and transcriptional activity of FOXM1 reduced upon RNF168 overexpression and increased with RNF168 depletion by siRNA, suggesting that RNF168 negatively regulates FOXM1 expression. Co-immunoprecipitation studies in MCF-7 cells revealed that RNF168 interacted with FOXM1 and that upon epirubicin treatment FOXM1 downregulation was associated with an increase in RNF168 binding and conjugation to the protein degradation-associated K48-linked polyubiquitin chains. Consistently, RNF168 overexpression resulted in an increase in turnover of FOXM1 in MCF-7 cells treated with the protein synthesis inhibitor cycloheximide. Conversely, RNF168, knockdown significantly enhanced the half-life of FOXM1 in both absence and presence of epirubicin. Using a SUMOylation-defective FOXM1-5x(K>R) mutant, we demonstrated that SUMOylation is required for the recruitment of RNF168 to mediate FOXM1 degradation. In addition, clonogenic assays also showed that RNF168 mediates epirubicin action through targeting FOXM1, as RNF168 could synergise with epirubicin to repress clonal formation in wild-type but not in FOXM1-deficient mouse embryo fibroblasts (MEFs). The physiological relevance of RNF168-mediated FOXM1 repression is further emphasized by the significant inverse correlation between FOXM1 and RNF168 expression in breast cancer patient samples. Moreover, we also obtained evidence that RNF8 recruits RNF168 to FOXM1 upon epirubicin treatment and cooperates with RNF168 to catalyse FOXM1 ubiquitination and degradation. Collectively, these data suggest that RNF168 cooperates with RNF8 to mediate the ubiquitination and degradation of SUMOylated FOXM1 in breast cancer genotoxic response.

The long-term outcome of high tibial osteotomy: a ten- to 20-year follow-up.

We carried out a prospective study of 132 patients (159 knees) who underwent closed-wedge high tibial osteotomy for severe medial compartment osteoarthritis between 1988 and 1997. A total of 94 patients (118 knees) was available for review at a mean of 16.4 years (16 to 20). Seven patients (7.4%) (11 knees) required conversion to total knee replacement. Kaplan-Meier survival was 97.6% (95% confidence interval 95.0 to 100) at ten years and 90.4% (95% confidence interval 84.1 to 96.7) at 15 years. Excellent and good results as assessed by the Hospital for Special Surgery knee score were achieved in 87 knees (73.7%). A pre-operative body mass index > 27.5 kg/m(2) and range of movement < 100 degrees were risk factors predicting early failure. Although our long-term results were satisfactory, strict indications for osteotomy are required if long-term survival is required.

The role of subchondral bone resorption pits in osteoarthritis: MMP production by cells derived from bone marrow.

OBJECTIVE: The vascular invasion of bone marrow tissue into the subchondral plate is often observed in articular cartilage and we named it the subchondral bone absorption pit; however, its implication in the pathogenesis of osteoarthritis (OA) has been poorly understood. The purpose of this study was to evaluate its characteristics and roles in osteoarthritic conditions. METHODS: Articular cartilage specimens from 11 patients with medial type knee OA and 7 non-arthritic cadavers were analyzed with HE staining. OA sections were stained with safranin-O, TRAP (tartrate resistant acid phosphatase) and immunostained with anti-MMP-1, MMP-3, MMP-13, vitronectin receptor (VNR)-alpha chain, vimentin and bone morphogenic protein (BMP) 2/4 antibodies. RESULTS: Subchondral bone resorption pits were classified according to the extent of invasion: pits with bone marrow tissue were located within uncalcified cartilage below the tidemark in grade I and invaded beyond the tidemark in grade II, while no invasion was seen in grade 0. Grade II pits were dominant in OA compared to non-arthritic joints, especially medial condyles. Proteoglycan detected with safranin-O staining was lost around the tip of grade II pits and the density of pits was related to the modified Mankin Score. Cells in pits expressed vimentin, MMP-1, MMP-3 and MMP-13. Some polynuclear cells co-expressed VNR-alpha chain and MMP-13, whereas pits showed reparative features expressing BMP. CONCLUSION: These results suggest that subchondral bone resorption pits contribute to cartilage degradation by expressing matrix metalloproteinases in OA.

Effects of glucosamine hydrochloride on the production of prostaglandin E2, nitric oxide and metalloproteases by chondrocytes and synoviocytes in osteoarthritis.

OBJECTIVES: To determine the response of glucosamine hydrochloride on chondrocytes and synoviocytes in terms of prostaglandin E2 (PGE2), nitric oxide (NO) and matrix metalloproteases (MMPs). METHODS: Chondrocytes and synoviocytes were prepared from joint specimens of patients who underwent total knee arthroplasty for osteoarthritis (OA). Chondrocytes from patients with femoral neck fracture were served as a normal control. Culture cells were stimulated by 5 ng/ml of IL-1beta and treated with various concentration of glucosamine hydrochloride (from 1 microg/ml to 500 microg/ml). PGE2, NO, MMP-1, MMP-3, and MMP-13 levels were evaluated in the culture supernatant. Further, the expression of COX-2 mRNA was studied by semiquantitative PCR. RESULTS: With IL-1beta stimulation, the levels of these mediators increased dramatically, except for NO from synoviocytes. After stimulation, levels of these mediators in OA chondrocytes were higher than synoviocytes and normal chondrocytes, and the level of MMP-3 was higher than those of MMP-1 and MMP-13. Glucosamine hydrochloride at a concentration of 100 microg/ml suppressed PGE2 production, and partly suppressed NO production. It also suppressed the production of MMPs from normal chondrocytes and synoviocytes but not from OA chondrocytes. CONCLUSION: Glucosamine modulates the metabolism of chondrocytes and synoviocytes and its mode of action differs between cells and conditions.

Characterization of cells from pannus-like tissue over articular cartilage of advanced osteoarthritis.

OBJECTIVE: To identify the characteristics of cells isolated from pannus-like soft tissue on osteoarthritic cartilage (OA pannus cells), and to evaluate the role of this tissue in osteoarthritis (OA). METHODS: OA pannus cells were isolated from pannus-like tissues in five joints obtained during arthroplasty. The phenotypic features of the isolated cells were characterized by safranin-O staining and immunohistochemical studies. Expression of MMP-1, MMP-3 and MMP-13 was also assessed using reverse transcriptase-polymerase chain reactions (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry. RESULTS: Foci and plaque formation of pannus-like tissue over cartilage surface were found in 15 of 21 (71.4%) OA joints macroscopically, and among them, only five samples had enough tissue to be isolated. OA pannus cells were positive for type I collagen and vimentin, besides they also expressed type II collagen and aggrecan mRNA. Spontaneous expression of MMP-1, MMP-3 and MMP-13 was detected in OA pannus cells. Similar or higher levels of MMPs were detected in the supernatant of cultured OA pannus cells compared to OA chondrocytes, and among these MMP-3 levels were relatively higher in OA pannus cells. Immunohistochemically, MMP-3 positive cells located preferentially in pannus-like tissue on the border of original hyaline cartilage. CONCLUSION: Our results showed that OA pannus cells shared the property of mesenchymal cells and chondrocytes; however, their origin seemed different from chondrocytes or synoviocytes. The spontaneous expression of MMPs suggests that they are involved in the articular degradation in OA.

Presence of pannus-like tissue on osteoarthritic cartilage and its histological character.

OBJECTIVE: To investigate and characterize pannus-like tissue which is often present on osteoarthritic articular cartilage. DESIGN: Cartilage specimens from 15 knee and five hip joints of patients with osteoarthritis (OA) undergoing arthroplasty were stained for HE and Safranin-O. They were also immunostained by antitype I collagen, type II collagen, CD68, IL-1beta and MMP3 antibodies. RESULTS: Ninety percent of joints have pannus-like tissue on the articular surface, preferentially in a marginal area. The articular cartilage was divided into three regions according to the location: the marginal zone, the intermediate zone and the paraeburnated zone. Pannuslike tissue in OA knee joint occurred 45.9%, 27.5% and 11.1% of the surface of each region respectively. Histologically, pannus-like tissue could be classified into the vascular type and the fibrous type. Extracellular matrix of both types of tissues were negative for Safranin-O and type II collagen, but positive for type I collagen. IL-1beta and MMP3 expressing cells are predominant in pannus-like tissue, whereas CD68 positive cells were infiltrated in only a few samples. Vascular type tissue kept continuity with bone marrow suggesting mesenchymal origin. CONCLUSION: Pannus-like tissue exists in advanced OA cartilage, preferentially in the marginal zone. It expressed IL-1beta and MMP3, which strongly suggests that it contributes to cartilage degradation.