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Differential Regulation of Proteoglycan 4 Metabolism in Cartilage by IL-1A, IGF-I, and TGF-B1 T

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Differential Regulation of Proteoglycan 4 Metabolism in Cartilage by IL-1A, IGF-I, and TGF-B1 T View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Osteoarthritis and Cartilage (2008) 16, 90e97 ª 2007 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.joca.2007.05.009 International Cartilage Repair Society Differential regulation of proteoglycan 4 metabolism in cartilage by IL-1a, IGF-I, and TGF-b1 T. A. Schmidt Ph.D., N. S. Gastelum B.S., E. H. Han M.S., G. E. Nugent-Derfus Ph.D., B. L. Schumacher B.S. and R. L. Sah M.D., Sc.D.* Department of Bioengineering and Whitaker Institute of Biomedical Engineering, University of California-San Diego, La Jolla, CA 92093-0412, United States Summary Objectives: To determine (1) if interleukin-1 alpha (IL-1a), insulin like growth factor I (IGF-I), and transforming growth factor-beta 1 (TGF-b1) regulate proteoglycan 4 (PRG4) metabolism in articular cartilage, in terms of chondrocytes expressing PRG4 and PRG4 bound at the articular surface, and (2) if these features of cartilage PRG4 metabolism correlate with its secretion. Methods: Articular cartilage explants were harvested and cultured for 6 days with or without 10% fetal bovine serum (FBS), alone, or with the addition of 10 ng/ml IL-1a, 300 ng/ml IGF-I, or 10 ng/ml TGF-b1. PRG4 expression by chondrocytes in the cartilage disks was assessed by immunohistochemistry (IHC). PRG4 bound to the articular surface of disks was quantified by extraction and enzyme-linked immunosorbent assay (ELISA). PRG4 secreted into culture medium was quantified by ELISA and characterized by Western Blot. Results: PRG4 expression by chondrocytes near the articular surface was markedly decreased by IL-1a, stimulated by TGF-b1, and not affected by IGF-I. The level of PRG4 accumulation in the culture medium was correlated with the number of chondrocytes expressing PRG4. The amount of PRG4 bound at the articular surface was modulated by incubation in medium including FBS, but did not correlate with levels of PRG4 secretion. Conclusions: Cartilage secretion of PRG4 is highly regulated by certain cytokines and growth factors, in part through alteration of the number of PRG4-secreting chondrocytes near the articular surface. The biochemical milieu may regulate the PRG4 content of synovial fluid during cartilage injury or repair. ª 2007 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Proteoglycan 4, Cartilage biology, Cytokines, Growth factors. Introduction Mutations of the PRG4 gene can cause camptodactyly- arthropathy-coxa vara-pericarditis (CACP) disease in Articular cartilage is a multifunctional tissue at the ends of humans17, which results in joint failure associated with long bones that allows for smooth articulation within dia- 1 non-inflammatory synoviocyte hyperplasia and subintimal rthrodial joints . Cartilage consists of three zones (super- fibrosis of the synovial capsule18. Both SZP and lubricin ficial, middle, and deep), each with distinct matrix 2,3 have boundary lubrication properties for native cartilage or structure and composition that impart specialized me- 9e14 4,5 certain synthetic surfaces . Since the Human Genome chanical properties to these regions . Chondrocytes of Organization Gene Nomenclature Committee assigned the middle and deep zones synthesize aggrecan at high the name PRG4 for the gene (GenBank accession no. rates, and thereby contribute to the ability of cartilage to AF056218 for bovine partial sequence and U70136 for com- bear compressive load. The chondrocytes of the superficial plete human sequence) encoding various immunoreactive zone exhibit a number of specific functions including the 8,17,19 6 proteins , including megakaryocyte stimulating factor, synthesis and secretion of superficial zone protein (SZP) . we refer collectively these molecules, herein, as w SZP is a 345 kDa protein present at the surface of PRG46,20. PRG4 therefore plays an important role in joint articular cartilage6, and in synovial fluid7, encoded for by 8 mechanics. the proteoglycan 4 (PRG4) gene . In joint injury and arthritis, the synovial fluid has impaired PRG4 molecules play an important role in the lubrication 21,22 9e14 friction-lowering lubrication qualities , possibly due to of articular cartilage by synovial fluid . In addition to en- altered in vivo metabolism of PRG4 under regulation of coding for SZP, the PRG4 gene encodes for a highly homol- the altered cytokine environment. In vitro studies have ogous protein termed lubricin15,aw220 kDa product of 16 demonstrated cytokines transforming growth factor-beta 1 synovial fibroblasts originally isolated from synovial fluid . (TGF-b1) and interleukin-1 alpha (IL-1a), which are present at elevated levels in joint injury and arthritis23, up-regulate *Address correspondence and reprint requests to: Dr Robert L. Sah, M.D., Sc.D., Department of Bioengineering, Mail Code 0412, and down-regulate PRG4 secretion into culture medium, re- 9500 Gilman Drive, University of California-San Diego, La Jolla, spectively, from superficial zone chondrocytes in explant CA 92093-0412, United States. Tel: 1-858-534-0821; Fax: 1-858- culture, as assessed by enzyme-linked immunosorbent as- 24 822-1614; E-mail: [email protected] say (ELISA) , and by chondrocytes in agarose culture, as Received 20 July 2006; revision accepted 14 May 2007. assessed by Western Blot19. Preliminary studies have 90 Osteoarthritis and Cartilage Vol. 16, No. 1 91 qualitatively characterized PRG4 expression to be regu- were viewed and documented by photomicroscopy to iden- lated in a similar manner, using immunohistochemistry to tify immunoreactive cells, indicating synthesis of PRG4. identify chondrocytes secreting PRG424. Insulin like growth The depth-associated variation in chondrocyte PRG4 ex- factor I (IGF-I), a potent physiological regulator of proteogly- pression was determined from these micrographs, essen- can metabolism by chondrocytes25,26, may also regulate tially as described previously30. Briefly, a representative PRG4 metabolism. These cytokines may also modulate 740 mm wide  300 mm deep region of each section was an- the amount of PRG4 bound at the surface of articular carti- alyzed using a custom program written in Matlab 6.5 software lage, where it is positioned to impart boundary lubrication (The Mathworks, Inc.). Chondrocytes expressing PRG4 function27. The relationships between the amount of (PRG4þ) were identified manually by one reader, in a blinded PRG4 bound at the articular surface, PRG4 expression manner. (This was confirmed as being reasonably indepen- within chondrocytes at or near the articular surface, and dent of the reader in these studies since eight micrographs PRG4 secretion from cartilage explants are unknown. Col- were also read by a second reader, and the number of lectively, these features of cartilage PRG4 metabolism PRG4þ cells identified were significantly correlated to those may be key aspects of PRG4 lubrication function. identified by the first reader, P < 0.001 and R2 ¼ 0.97.) Then, The objectives of this study were therefore to determine the total number of PRG4þ cells, and the number of PRG4þ (1) if IL-1a, IGF-I, and TGF-b1 regulate PRG4 metabolism cells as a function of depth from the articular surface were in articular cartilage, in terms of chondrocytes expressing counted. Results are presented as the number of PRG4þ PRG4 and PRG4 bound at the articular surface, and (2) if cells in successive 50 mm bins below the articular surface. these features of cartilage PRG4 metabolism correlate The total number of cells counted in each section was with its secretion. 300 Æ 98 (mean Æ standard deviation (SD). PRG4 surface concentration Methods PRG4 bound to the articular surface of freshly isolated MATERIALS disks, and those cultured for 6 days, was determined by ex- 31 Materials for tissue harvest and culture were obtained as traction and ELISA, as described previously . Briefly, four described previously24,28. Recombinant human IL-1a, por- 3 mm disks were nutated in 0.4 ml of 4 M GuHCl, 0.02 M cine TGF-b1, and recombinant human IGF-I were obtained Tris, pH 8.2 containing protease inhibitors (0.005 M benza- from R&D Systems (Minneapolis, MN). midine HCl, 0.01 M N-ethylmaleimide, 0.005 M disodium ethylenediamine tetraacetic acid , and 0.001 M phenylme- thylsulfonylfluoride) for 24 h. Portions (200 ml) of extracts CARTILAGE EXPLANT HARVEST AND CULTURE or purified bovine standards6 were then applied to a nitrocel- lulose membrane in a Bio-Dot apparatus (Bio-Rad, Hercu- Cartilage explants were harvested and cultured as les, CA), blocked with 5% non-fat dry milk, incubated with described previously24. Briefly, osteochondral cores were mAb 3A4, and then with an anti-mouse antibody conjugated harvested from the patellofemoral groove of immature to horseradish peroxidase, with rinses in PBS between (1e3 week old) bovine stifle joints. These cores were cut each step. The sample areas were removed and reacted in a sledge microtome to obtain slices (0.3 mm thick) con- with 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) taining the superficial zone, with the articular surface intact. (ABTS) substrate. Control studies, where a known amount These slices of cartilage were then punched into smaller of PRG4 (in the form of medium conditioned by explants 3 mm diameter disks. Disks were then incubated for 6 containing the superficial zone and quantified by ELISA) days in a basal medium (Dulbecco’s modified Eagle me- was added to serial dilutions of sample
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