A Novel Gene Coding for a Fas Apoptosis Inhibitory Molecule (FAIM) Isolated from Inducibly Fas-resistant B Lymphocytes

Abstract

The sensitivity of primary splenic B cells to Fas-mediated apoptosis is modulated in a receptor-specific fashion. Here we used a differential display strategy to detect cDNAs present in B cells rendered Fas resistant but absent in those rendered Fas sensitive. This led to the cloning and characterization of a novel 1.2-kb gene that encodes a Fas apoptosis inhibitory molecule (FAIM). faim-transfected BAL-17 B lymphoma cells were less sensitive by half or more to Fas-mediated apoptosis than were vector-transfected controls, using Fas ligand–bearing T cells or a cytotoxic anti-Fas antibody to trigger Fas, and this was associated with inhibition of Fas- induced poly-ADP ribose polymerase (PARP) cleavage. In primary B cells, the time course of faim mRNA and FAIM protein expression correlated with the induction of Fas resistance by surface (s)Ig engagement. Thus, FAIM is an inducible effector molecule that mediates Fas resistance produced by sIg engagement in B cells. However, faim is broadly expressed in various tissues and the faim sequence is highly conserved evolutionarily, suggesting that its role extends beyond lymphocyte homeostasis. As FAIM has no significant regions of homology to other gene products that modulate Fas killing, it appears to represent a distinct, new class of antiapoptotic protein.

Inducible resistance to Fas-mediated apoptosis in B cells

“…Along the same lines, FAIM protein increased within 6 h of treating CD40L-stimulated B cells with anti-Ig, and expression continued to rise, peaking at 12 h, as detected by Western blotting [51]. The finding of substantially increased FAIM expression in splenocytes obtained from sheep red blood cell-immune animals, as compared to splenocytes obtained from control animals injected with saline, suggests that these in vitro studies of inducible FAIM expression bear relevance for physiological immune responses.…”

“…To examine this possibility, B cells were treated with inhibitors of macromolecular synthesis along with anti-Ig during the last 6 h of 48 h cultures with CD40L (longer periods of exposure being precluded by drug toxicity); the addition of either cycloheximide or actinomycin D completely blocked the induction of Fas-resistance by anti-Ig [35], [51]. Thus, Fasresistance induced by sIg engagement appears to depend on new gene expression and new protein synthesis.…”

“…Through this approach, a completely novel 1.2 kb sequence was cloned that contains an open reading frame encoding a protein product of 179 amino acids predicted to reflect a stable, soluble, β-strand-rich molecule that is slightly acidic [51]. The function of this gene was evaluated with BAL-17 murine B lymphoma cells, the activation responses of which mimic those of primary B cells in a number of ways [92], including upregulated Fas expression and increased sensitivity to Fas killing following CD40L stimulation.…”

“…Cytotoxicity was assessed in two ways, by chromium release after exposure of transfectants to FasL-bearing Th1 effector cells, and by propidium iodide-staining for subdiploid DNA after treatment of transfectants with Jo-2 anti-Fas antibody. Regardless of the means by which Fas was engaged or cytotoxicity was assessed, Fas-mediated apoptosis was markedly reduced in B cells expressing the gene of interest as compared to vector transfectants, using both G418-resistant pools and stably transfected lines cloned by limiting dilution [51]. Considering the dose-response curve with Th1 effector cells, Fas-resistance produced by the gene of interest amounted to a greater than 9-fold level of protection against Fas-mediated apoptosis.These results indicate that the differentially expressed gene encodes a Fas Apoptosis Inhibitory Molecule, which we termed FAIM.…”

“…The anti-apoptotic function of FAIM was demonstrated further at a biochemical level by evaluating cleavage products of poly-ADP ribose polymerase (PARP) that result from caspase activation [93,94]. PARP fragments were detected within 40 minutes of treating vector-transfected BAL-17 B cells with Jo-2 anti-Fas antibody, whereas such fragments were not observed in faim-transfected BAL-17 B cells up to 60 minutes after antibody treatment [51]. This correlated well with the transgenically enhanced level of FAIM expression, which was demonstrated by Western blotting to be greatly increased over the endogenous levels present in vector-transfected controls.…”