Rheumatoid arthritis (RA) is currently considered an autoimmune disease in which a pathologic immune response attacks synovial cells, cartilage and bone resulting in joint destruction and permanent disability. Loss of bone mass in RA is a common clinical problem occurring as both juxta-articular or localized bone loss and generalized osteoporosis leading to the risk of fracture. The juxta-articular bone loss is associated with an increase in blood- derived T cells infiltrating into the synovial cavity. Activated (Act) T cells are believed to mediate most of the tissue destruction, once the inflammation cascade has begun. The Act T cell is a source of cytokines known to regulate bone turnover, such as IL-6 , TNF-alpha and a new member of the TNF receptor family, TRANCE/RANKL/ODF/OPGL, but a causative T cell cytokine is presently unknown. This project is aimed at discovering whether a cytokine produced by activated T cells, that we have recently found regulates the expression of IL-6 in normal human osteoblasts and regulates osteoclast differentiation, is a novel protein. The identification of a novel T cell factor which may be inducing osteopenia in rheumatoid arthritis and perhaps other autoimmune diseases may lead to the development of inhibitory agents which may prove to be of therapeutic efficacy in preventing bone loss in these diseases. Thus our specific Aims are: To purify, characterize and clone the soluble factor(s) produced by activated T-cells that regulates IL-6 production in human osteoblasts and induces osteoclastogenesis; and 2) Prepare recombinant protein and produce antibodies to the T cell factor for future studies of its function. The factor(s) will be isolated using affinity chromatogphy, anion and cation exchange chromatography, gel filtration chromatography and SDS-PAGE. Identification of the proteins as novel will be performed by N-terminal or internal sequencing. Internal sequence synthetic peptides will be synthesized and used for the production of polyclonal antibodies. Cloning will be performed using the rapid amplification of cDNA ends (RACE) technique to obtain a partial human cDNA from adaptor-ligated human T cell cDNA. Primers will be designed and the full length cDNA reading frame will be PCR amplified, primers prepared and ultimately a fusion protein cDNA prepared and ligated into a prokaryotic expression vector in order to produce recombinant protein. Antibodies will be prepared against the protein to characterize its function in vitro. We propose to determine whether the T cell factor induces TRANCE in hOB by probing isolated mRNA with labeled cDNA, generated by RT-PCR methods, and Northern blot analysis.
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| Rifas, Leonard; Arackal, Sophia (2003) T cells regulate the expression of matrix metalloproteinase in human osteoblasts via a dual mitogen-activated protein kinase mechanism. Arthritis Rheum 48:993-1001 |
| Rifas, Leonard; Cheng, Su-Li (2003) IL-13 regulates vascular cell adhesion molecule-1 expression in human osteoblasts. J Cell Biochem 89:213-9 |
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| Weitzmann, M N; Cenci, S; Rifas, L et al. (2000) Interleukin-7 stimulates osteoclast formation by up-regulating the T-cell production of soluble osteoclastogenic cytokines. Blood 96:1873-8 |
