Cloning of follistatin-related protein as a novel autoantigen in systemic rheumatic diseases

Abstract

In an attempt to identify autoantigens of synovium in rheumatoid arthritis (RA), we constructed lambda phage expression cDNA libraries from synovium and screened them by IgG purified from synovial fluids, both of which were derived from RA patients. As a result of this unique combination of the libraries and probes, we cloned follistatin-related protein (FRP) as a novel autoantigen in systemic rheumatic diseases. FRP is a secreted protein containing a similar amino acid sequence to follistatin, an inhibitor of activin. FRP was first cloned as a transforming growth factor-beta1-inducible protein (called TSC-36) from a mouse osteoblastic cell line and was suggested to have some roles in the negative regulation of cellular growth. Immunoblotting analyses detected synovial fluid and serum anti-FRP antibodies of IgG class more frequently in RA than any other systemic rheumatic diseases and controls. Synovial fluid anti-FRP antibodies appeared in 44% of RA (n = 18) and none of osteoarthritis (OA) (n = 15) patients. Serum antibodies were detected in 30% of RA (n = 67), 17% of systemic sclerosis (n = 18), 10% of systemic lupus erythematosus (n = 51) and Sjögren's syndrome (n = 10), and none of polymyositis/dermatomyositis (n = 13) patients and healthy subjects (n = 30). These antibodies recognized an EC domain, an extracellular Ca2+ binding module. In anti-FRP antibody-positive RA patients, serum C-reactive protein level and erythrocyte sedimentation rate were more elevated than negative patients (P < 0.05 and P < 0.01, respectively). FRP gene expression was higher in RA than OA synovium (P < 0.05). However, there was no difference between these groups in the amount of synovial FRP, suggesting its elevated turnover in RA. As follistatin inhibits activin, FRP might inhibit some growth factor-like molecule. Detection of anti-FRP antibodies, possibly having disease-promoting effects as the blocking antibodies, could be one of the markers for clinical evaluation of systemic rheumatic diseases.

Ameliorative effects of follistatin‐related protein/TSC‐36/FSTL1 on joint inflammation in a mouse model of arthritis

“…We previously reported that serum autoantibodies to FRP were detected in 30% of patients with RA, and their appearance correlated with disease activity (7). Recently, our in vitro experiments demonstrated that FRP suppressed the production of matrix metalloproteinase 1 (MMP-1), MMP-3, and prostaglandin E 2 in synovial cells derived from patients with RA, and suggested that FRP could act as a joint defensive factor in RA (17).…”

“…Escherichia coli-expressed glutathione S-transferase (GST)-FRP was prepared as previously described (7). The GST tag was removed from GST-FRP using a GSTrap FF column (Amersham Pharmacia Biotech, Uppsala, Sweden), and the protein was purified by an anionexchange chromatography method with a Mono Q HR column (Amersham Pharmacia Biotech).…”

Expression, characterization, and preliminary X-ray crystallographic analysis of recombinant murine Follistatin-like 1 expressed in Drosophila S2 cells

“…Previous reports have described the expression and purification of recombinant FSTL1 (12,23,24,27). E. coli strains were used as the expression host in some of these reports; however, in some of these studies, the expressed recombinant FSTL1 protein had less biological activity due to incomplete post-translational modification of FSTL1 in the prokaryotic system (12,24,27).…”

“…In vivo, FSTL1 is expressed temporally and spatially and is generally associated with tissues undergoing remodeling, either during normal developmental processes or in response to injury. Evidence has implicated FSTL1 in a number of pathologic conditions, including inflammation (11), rheumatoid arthritis (12)(13)(14)(15)(16), tumorigenesis (17)(18)(19), and heart disease (20,21). Functions of FSTL1 in the extracellular milieu are diverse and remain elusive.…”

Inhibition of cartilage destruction by double gene transfer of IL-1Ra and IL-10 involves the activin pathway

“…72 IL-4 mediated growth arrest and IL-10 mediated inhibition of proliferation involve also p21CIP1, 73,74 revealing additional effector mechanisms of vIL-10 and IL-1Ra double gene transfer (Figure 7). Moreover, follistatin-related protein (FRP), which contains a similar amino acid sequences to follistatin, and is supposed to be an autoantigen in systemic rheumatic diseases, 75 was also down-regulated following double gene transfer of IL-1Ra and IL10.…”