Introduction
Rheumatoid arthritis (RA) has a prevalence of about 1% in most parts of the world. While targeting TNFα using biological inhibitors has been an undoubted success, efficacy does not usually approach remission. Moreover, increasing usage of anti-TNFα biological agents in RA is associated with an augmented risk of infections, including tuberculosis [1-5]. As a consequence, initiatives to develop alternative targets in RA are desirable, especially for use in combination with TNFα inhibitors.
Cell surface receptors such as receptor tyrosine kinases (RTKs) mediate ligand-induced signal transduction from the extracellular to the intracellular environment. Dysregulation of RTK signaling is implicated in the pathogenesis of many human diseases, including cancer and autoimmune diseases [6,7]. The discovery that soluble forms of receptors can abrogate receptor–ligand interaction has fueled substantial interest in their potential application as biotherapeutics. Etanercept, a molecularly engineered fusion protein composed of the extracellular domain of TNF receptor type II, is an example of a clinically effective soluble receptor-based therapeutic, with potent activity in RA [8].
Soluble receptors are known to occur naturally in vivo [9]. Two major mechanisms involved in the formation of naturally occurring soluble receptors are proteolytic cleavage of membrane receptors and alternative pre-mRNA splicing. The latter is a process in which multiple proteins are created from a single pre-mRNA [10-13]. Bioinformatics analyses predict that the majority of human genes undergo alternative splicing, suggesting that alternative splicing is a significant component in generating diversity of function in the human genome [11]. The protein products of alternative splicing may serve as homeostatic regulators in physiology and disease [14-16]. This is illustrated by the splice variant of vascular endothelial growth factor receptor (VEGFR) type 1 (sVEGFR1 or sFlt-1). Vascular endothelial growth factor (VEGF) plays a pivotal role in regulating angiogenesis, and binds sFlt-1 in vivo. Suppression of endogenous sFlt-1 was found to abolish corneal avascularity in mice [17]. Conversely, sFlt-1 has been shown to modulate disease in other in vivo models, including animal models of RA [18-22].
To determine the frequency of functional soluble splice forms of cell surface receptors, we have developed a high-throughput method for gene scanning, cloning, and characterization that identified functional alternative splice variants (ASV). The present work describes the RT-PCR selection and molecular cloning of 60 novel soluble receptors as splice variants of 21 RTKs and other cell surface receptor genes, including VEGF and TNF receptors. These cell surface receptor-derived ASV differ from transmembrane proteins, or shed receptors, by the deletion or addition of unique amino acids as a result of alternative splicing events, including exon extensions and deletions. The novel ASV that we identified included splice variants of receptors for VEGF (VEGFR1, VEGFR2 and VEGFR3) and for angiopoietin-1 (Ang-1) receptor Tie1 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 1), as well as for platelet-derived growth factor receptor beta (PDGFRβ) and fibroblast growth factor receptors (FGFRs).
We selected 10 ASV for further analysis, chosen on the basis of their potential effects on angiogenesis, which represent an attractive target for therapy in RA [23-28]. We confirmed that ASV derived from cell surface receptors retained their ligand binding ability and were transcribed in human normal and malignant tissues. Furthermore, using adenoviruses expressing secreted ASV, we demonstrated that these ASV exhibit differential effects in a murine model of RA – namely, collagen-induced arthritis (CIA), which is in widespread use as a tool for developing new therapeutics. Work in the acute CIA model formed the basis for the widespread clinical use of TNFα inhibitors for treatment of RA [29-32]. Moreover, we and other workers have shown that inhibition of angiogenesis ameliorates disease [18,20,33-38]. We observed that ASV corresponding to VEGFR1, and to a lesser extent VEGFR2, reduced arthritis severity, in agreement with our earlier findings using sFlt-1 [18,20]. We also observed for the first time that ASV corresponding to Tie1 significantly reduced arthritis severity and joint destruction. While expression of Ang-1 [39,40] and of Tie receptors [41-43] has been reported in RA, this is the first demonstration that Tie1 is effective in an in vivo model of arthritis. We also observed a modest effect of FGFR1 ASV in acute CIA.
These data establish that ASV derived from receptors that play key roles in angiogenesis – VEGFR1 and, for the first time, Tie1 – can reduce arthritis severity. More broadly, ASV are a source of novel proteins with therapeutic potential in diseases in which angiogenesis and cellular hyperplasia play a central role, such as RA.