Our research goal is to understand the biological role of terminal complement complexes (C5b-7, C5b-8, C5b-9) in inflammation and immune reaction. We and others have shown that eukaryotic cells are protected from the cytolytic C5b-9 h species-specific restriction of C5b-9 assembly and via elimination of membrane-inserted TCC. Cells exposed to limited C5b-9 are therefore survive, but with cell phenotype alteration. Sublytic C5b-9 acts as a mitogen, and this is shown by induction of proto-oncogenes and increased DNA synthesis with cell growth. The Ras, Raf-1, and ERK1 pathway is activated by TCC. and this is one pathway transducing signaling to nucleus. We have cloned a novel gene RGC-32 (Response Gene to Complement), which is activated by C5b-9 and involved in cell cycle. RGC-32 overexpression increased DNA synthesis. RGC-32 complexes with cdc2 in cells, which is increased by C5b-9. In cell-free systems, the cdc2 kinase activity is markedly increased by RGC-32. In terminally differentiated cells such as oligodendrocytes, C5b-9 increases cell survival without cell growth, through inhibition of apoptosis. Our preliminary data indicated both pro- and anti-apoptotic BCL-2 family proteins are regulated by C5b-9. These novel functions of C5b-9 may be involved in cell phenotype alteration during inflammation and immune reactions. Two specific hypotheses are proposed.
Aim 1. RGC-32 is involved in cell cycle activation through regulation of cell cycle kinases. To test this, we will examine (i) DNA synthesis and cell growth affected by overexpression of RGC-32 and by inhibition of endogenous RGC-32, (ii) the binding to and activation of cdc2 by using mutated and deleted recombinant RGC-32 proteins, and (iii) the role of TCC signaling on the activity of RGC-32.
Aim 2. C5b-9 increases survival of oligodendrocytes by inhibiting mitochondrial pathway apoptosis. This hypothesis will be explored by identifying the steps of mitochondrial pathway apoptosis affected by C5b-9, and by investigating C5b-9-mediated regulation of the pro- and anti-apoptotic BCL-2 family of proteins.
