Available online http://arthritis-research.com/content/7/2/R274 ResearchVol 7 No 2 article Open Access Repression of anti-proliferative factor Tob1 in osteoarthritic cartilage Mathias Gebauer1*, Joachim Saas2*, Jochen Haag3, Uwe Dietz2, Masaharu Takigawa4, Eckart Bartnik2 and Thomas Aigner3 1Aventis Pharma Deutschland, Functional Genomics, Sanofi-Aventis, Frankfurt, Germany 2Sanofi-Aventis, Disease Group Thrombotic Diseases/Degenerative Joint Diseases, Frankfurt, Germany 3Osteoarticular and Arthritis Research, Department of Pathology, University of Erlangen-Nürnberg, Germany 4Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan * Contributed equally Corresponding author: Thomas Aigner, [email protected] Received: 10 Aug 2004 Revisions requested: 1 Oct 2004 Revisions received: 22 Oct 2004 Accepted: 19 Nov 2004 Published: 11 Jan 2005 Arthritis Res Ther 2005, 7:R274-R284 (DOI 10.1186/ar1479)http://arthritis-research.com/content/7/2/R274 © 2005 Gebauer et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Osteoarthritis is the most common degenerative disorder of the genes were detected between normal and osteoarthritic modern world. However, many basic cellular features and cartilage (P < 0.01). One of the significantly repressed genes, molecular processes of the disease are poorly understood. In Tob1, encodes a protein belonging to a family involved in the present study we used oligonucleotide-based microarray silencing cells in terms of proliferation and functional activity. analysis of genes of known or assumed relevance to the cellular The repression of Tob1 was confirmed by quantitative PCR and phenotype to screen for relevant differences in gene expression correlated to markers of chondrocyte activity and proliferation in between normal and osteoarthritic chondrocytes. Custom made vivo. Tob1 expression was also detected at a decreased level in oligonucleotide DNA arrays were used to screen for differentially isolated chondrocytes and in the chondrosarcoma cell line expressed genes in normal (n = 9) and osteoarthritic (n = 10) HCS-2/8. Again, in these cells it was negatively correlated with cartilage samples. Real-time polymerase chain reaction (PCR) proliferative activity and positively with cellular differentiation. with gene-specific primers was used for quantification. Primary Altogether, the downregulation of the expression of Tob1 in human adult articular chondrocytes and chondrosarcoma cell osteoarthritic chondrocytes might be an important aspect of the line HCS-2/8 were used to study changes in gene expression cellular processes taking place during osteoarthritic cartilage levels after stimulation with interleukin-1β and bone degeneration. Activation, the reinitiation of proliferative activity morphogenetic protein, as well as the dependence on cell and the loss of a stable phenotype are three major changes in differentiation. In situ hybridization with a gene-specific probe osteoarthritic chondrocytes that are highly significantly was applied to detect mRNA expression levels in fetal growth correlated with the repression of Tob1 expression. plate cartilage. Overall, more than 200 significantly regulated Keywords: bone morphogenetic protein, cartilage, chondrocytes, gene expression, proliferation Introduction in [2]). First, the chondrocytes can degenerate or prolifer- Osteoarthritis is the most common disabling condition of ate. Second, chondrocytes can activate or deactivate their humans in the western world. Although osteoarthritis is synthetic anabolic or catabolic matrix-degrading activity by mainly a disease and functional loss of the articular carti- increasing or decreasing anabolic or catabolic gene lage covering the joint surfaces, it is clearly the cells that are expression. Last, chondrocytes can undergo phenotypic the active players during the disease process [1]. What- modulations implicating an overall severely altered gene ever pleomorphisms the cellular reaction patterns display at expression profile of the cells in the diseased tissue. In fact, first sight during the osteoarthritic disease process, they several distinct phenotypes of chondrocytes are known to can be basically summarized in three categories (reviewed occur in vitro, in vivo during fetal development and BMP = bone morphogenetic protein; cDNA = complementary DNA; cRNA = complementary RNA; IL = interleukin; MMP = matrix metalloproteinase; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; qPCR = quantitative polymerase chain reaction; UTR = untranslated region. R274 Arthritis Research & Therapy Vol 7 No 2 Gebauer et al. potentially also in the disease process itself, but new mark- Isolation of primary human articular chondrocytes; ers are required for the more accurate characterization of stimulation with interleukin (IL)-1β and bone cellular behavior [3]. This will allow further analysis of the morphogenetic protein (BMP)-7 underlying pathology to develop therapeutic approaches Normal human knee articular cartilage was obtained from that could delay, stop, or even reverse cartilage six normal cases at autopsy within 48 hours of death. Car- degeneration. tilage pieces were finely chopped and chondrocytes were isolated enzymatically as described previously [7]. In many laboratories single and multiple gene analyses Chondrocytes were either plated in high-density monolayer have been performed on normal and osteoarthritic cartilage cultures or cultured in alginate beads. Cultures were main- specimens; however, a global overview of disease-associ- tained for 48 hours in serum-free Dulbecco's modified ated changes is not available. This highlights the need for Eagle's medium/F12 medium (Gibco BRL, Eggstein, Ger- establishing a broader gene expression profile of osteoar- many) supplemented with 1% penicillin/streptomycin solu- thritic chondrocytes by modern screening technologies so tion (Gibco BRL) and 50 µg/ml ascorbate (Sigma, as to characterize more properly the cellular events and Taufkirchen, Germany) and 10% fetal calf serum (Bio- regulatory pathways directly involved in cartilage destruc- chrom, Berlin, Germany). tion. In the present study, we designed a custom-made oli- gonucleotide-based microarray to screen for differentially After 48 hours, primary (non-passaged) chondrocytes were expressed genes in normal and osteoarthritic cartilage stimulated with 1 ng/ml IL-1β (R&D System, Minneapolis, specimens. We found that Tob1, a gene involved in cell MN, USA) in DMEM/F12 medium, 100 ng/ml recombinant cycle regulation and cell quiescence [4,5], was significantly human BMP-7 (Stryker Biotech, Hopkinton, MA, USA) or repressed in osteoarthritic chondrocytes. This was con- cultivated in medium alone for 24 hours with no medium firmed by quantitative polymerase chain reaction (qPCR) change afterwards. The same experiments were performed and further analyzed in adult articular chondrocytes in vitro in parallel in the presence and in the absence of 10% fetal and in vivo. calf serum. At the end of the culturing/stimulation period the cells were washed in sterile phosphate-buffered saline Materials and methods (PBS), lysed in 350 µl of lysate RLT buffer/106 cells and Donors for mRNA expression analysis stored at -80°C. For the study of mRNA expression levels within the tissue, cartilage from human femoral condyles of normal knee Culture of HCS-2/8 cells joints was used. Normal articular cartilage (nqPCR = 10, age The human HCS-2/8 chondrosarcoma cell line (around range 45–88 years, mean age 64.1 years; narray = 9, age passage 50–55) [8,9] was cultured in DMEM (PAA, Linz, range 37–83 years, mean age 59 years) was obtained from Austria) supplemented with 20% fetal bovine serum (Gibco donors at autopsy, within 48 hours of death. Osteoarthritic BRL) and with 50 µg/ml ascorbate (Sigma) in a humidified cartilage samples from late-stage osteoarthritic joint dis- atmosphere of 5% CO2 at 37°C as described [9]. Cells ease were obtained from patients undergoing total knee were seeded at 105 cells/cm2 and grown for 3 days to 5 2 replacement surgery (nqPCR= 15, age range 63–85 years, obtain subconfluent stage cultures, at 2 × 10 /cm and cul- mean age 74.5 years; narray = 10, age range 57 to 84 years, tured for 7 days to obtain confluent stage cultures, and at mean age 76 years). The cartilage was frozen in liquid nitro- 6 × 105/cm2 and grown for 10 days for over-confluent gen immediately after removal and stored at -80°C until stage cultures. required for RNA isolation. RNA isolation from articular cartilage and isolated Cartilage was considered to be normal according to a mac- articular chondrocytes roscopic scoring system of the opened joint: this mainly Total RNA from both cartilage tissue and isolated chondro- included normal synovial membrane, normal synovial fluid, cytes was isolated as described previously [10,11]. The no significant overall softening or surface fibrillation (except quality of total RNA samples was checked by agarose-gel on the tibial plateau, which is basically found in all speci- electrophoresis and with the Bioanalyzer RNA 6000 Nano mens depending on age). The Mankin's grade of histologi- assay (Agilent, Waldbronn, Germany). cal plugs taken was less than