A neuronal C5a receptor and an associated apoptotic signal transduction pathway

1998 
We report the first experimental evidence of a neuronal C5a receptor (nC5aR) in human cells of neuronal origin. Expression of nC5aR mRNA was demonstrated by the reverse transcriptase-polymerase chain reaction (RT-PCR) in TGW human neuroblastoma cells. Expression of a functional C5aR was supported by the finding that C5a evoked a transient increase in the intracellular calcium level as measured by flow cytometry (FACS). To analyse the function of the nC5aR, an antisense peptide fragment of the C5aR was used. Previous data showed that a C5aR fragment (a peptide termed PR226) has C5aR agonist and antagonist effects in U-937 cells depending on the concentration of the peptide. We found that a multiple antigenic peptide (MAP) form of the same peptide (termed PR226-MAP) induced rapid elevation of nuclear c-fos immunoreactivity and resulted in DNA fragmentation, a characteristic sign of apoptosis, in TGW cells. Early electrophysiological events characteristic of apoptosis were also detected: intermittent calcium current pulses were recorded within 1–2 min of peptide administration. C5a pretreatment delayed the onset of this calcium influx. We also demonstrated that the apoptotic pathway is linked to nC5aR via pertussis toxin-sensitive G-proteins. Although the function of C5a and its receptor on neurons is unknown, these results suggest that an abnormal activation of this signal transduction pathway can result in apoptosis and, subsequently, in neurodegeneration. Activation of the complement system is accompanied by the release of a seventy-four amino acid long amino terminal fragment of the complement factor C5. This fragment, C5a, is one of the most potent mediators of inflammation, acts as a chemoattractant for leukocytes and activates phagocytosis (reviewed by Rother & Till, 1988). The C5a receptor (C5aR) is a member of the rhodopsin superfamily of proteins bearing seven transmembrane regions and it is associated with both pertussis toxin-sensitive and -resistant G-proteins (Amatruda, Steele, Slepak & Simon, 1991; Gerard & Gerard, 1991). Earlier, expression of C5aR was reported in leukocytes; however, it has also been demonstrated in other cell types, such as liver parenchymal cells, lung bronchial and alveolar epithelial cells, vascular smooth muscle and endothelial cells as well as in human astrocytes (Huey & Hugli, 1985; Gerard, Hodges, Drazen, Weller & Gerard, 1989; Gasque et al. 1995; Havilland et al. 1995). In this report, we demonstrate for the first time that cells of a TGW human neuroblastoma cell line express a C5aR-like molecule termed neuronal C5aR (nC5aR). In order to analyse the function of C5aR, antisense homology box peptides were generated to modify the activity of the receptor. Antisense homology boxes (AHBs) in C5aR and its ligand C5a have been analysed recently (Baranyi, Campbell & Okada, 1996). AHBs are relatively short eight to fifteen amino acid long regions in proteins related in a sense-antisense fashion as if encoded by complementary DNA strands and have been found to be hydropathically complementary. The peptides corresponding to complementary AHBs are predicted to interact (Baranyi, Campbell, Ohshima, Fujimoto, Boros & Okada, 1995). One of the peptides representing an AHB in C5aR (amino acids 226–243, termed PR226) was synthesized and found to induce a C5aR-specific agonist effect when administered at low doses or alternatively behaves as an antagonist at high doses and influences C5a binding in U-937 cells (Baranyi et al. 1996). We used the oligomeric (multiple antigenic peptide, MAP) form of the PR226 peptide (Peptide8-K4K2K, termed PR226-MAP) to modulate C5aR-specific responses in cells since the MAP form of peptides has been shown to be more potent than the monomers of the respective peptides (Fassina, Consonni, Zetta & Cassani, 1992). Furthermore, the PR226MAP peptide was shown to be a potent inhibitor of C5aR, while at high doses it induced apoptotic cell death in U-937 cells (L. Baranyi, W. Campbell, T. Soji, I. Farkas, K. Baranji, N. Okada & H. Okada, unpublished data).
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