Rat FAIM cloning and expression
FAIM is a newly identified antiapoptotic protein that is highly conserved among multiple animal phyla and is expressed in at least two isoforms, long and short (Schneider et al., 1999
, Zhong et al., 2001
). By searching publicly available EST databases (GenBank, NCBI, and NIH), we found clones encoding both the short and the long forms of rat FAIM. A compares the FAIM sequences from Rattus norvegicus
with Drosophila melanogaster
, C. elegans
, Gallus gallus
, Danio rerio
, Mus musculus
, and Homo sapiens
. The cloned rat sequences are available from GenBank/EMBL/DDBJ under accession no. NP_543171
(short form) and AAL77007
(long form). Alignment of FAIM vertebrate sequences shows more than 68% of homology. The percentage of homology is reduced to 30–50% when vertebrate sequences were aligned with D. melanogaster
and C. elegans
FAIM sequences, respectively ().
Figure 1. Protein alignments of FAIM. (A) Comparison of FAIM protein sequences from rat, mouse, human, C. elegans, D. melanogaster, G. Gallus, and D. rerio. Identical amino acids are shaded in black, and those with conservative changes in gray. The additional 22 (more ...)
Percentage of identity in a pairwise alignment between different species
Using RT-PCR, we found that FAIM mRNA is expressed in a wide variety of tissues including all the neural tissue samples examined (cortex, cerebellum, hippocampus, hindbrain, and spinal cord; B). FAIM is also expressed in PC12 cells, mouse superior cervical ganglion (SCG) neurons, and cortical neurons ( B).
FAIM fails to protect neurons from neurotrophic factor deprivation but increases NGF-induced neurite outgrowth
NGF induces survival and neurite outgrowth from certain peripheral nervous system neurons and promotes the differentiation of PC12 cells to neuronal-like cells with neuritic processes. PC12 cells and SCG neurons require NGF for in vitro survival, and NGF withdrawal is rapidly followed by cell death. Because FAIM was initially cloned as an antagonist of Fas, and because Fas has been implicated in some types of neuronal cell death, we wanted to ascertain if FAIM was able to prevent cell death after NGF deprivation. To this end, we generated pools of PC12 cells stably transfected with either FAIM or the corresponding empty vector (Neo). These cells were grown in the presence of NGF for 72 h, and then were deprived of NGF for 24 h and cell viability was assessed. NGF-deprived, FAIM-transfected PC12 cells showed a reduction in survival (55.1 ± 8.0% survival) similar to that observed in NGF-deprived Neo cells (46.2 ± 6.4% survival). Similar results were obtained using mouse SCG neurons. For these experiments, SCG neurons were transiently transfected with FAIM or the empty vector and were grown with NGF for 24 h before NGF deprivation, and the number of surviving neurons was counted 24 h later. NGF-deprived, FAIM-transfected SCG showed a dramatic decrease in cell viability (8.1 ± 2.3% survival) similar to that found in NGF-deprived empty vector-transfected neurons (6.1 ± 1.4% survival). As a positive control, we transfected SCG cultures with Bcl-XL, a well characterized antiapoptotic molecule of the Bcl-2 family. Neurons overexpressing Bcl-XL survived NGF deprivation (81.6 ± 10.9% survival). These results demonstrate that FAIM is unable to prevent cell death after NGF deprivation in both PC12 and SCG neurons.
Despite the absence of an antiapoptotic function in PC12 cells and SCG neurons, we noticed that when these cells were transfected with FAIM and maintained in presence of NGF, there was a marked increase in neurite outgrowth compared with empty vector-transfected neurons. To ascertain if FAIM was able to modulate NGF-induced neurite outgrowth or influence neurite growth independently of NGF, pools of PC12 cells stably transfected with FAIM or the empty vector were grown with or without NGF for 1 d. shows that in NGF-supplemented medium, PC12 cells overexpressing FAIM exhibited a fivefold increase in neurite length compared with control-transfected cells. However, overexpression of FAIM did not have any effect on neurite outgrowth from PC12 cells grown in complete medium without NGF. These results suggest that FAIM is able to regulate neurite outgrowth in NGF-differentiated PC12 cells. When the long form of FAIM was tested in the same assay, no enhanced neurite outgrowth was observed () and, therefore, the studies using this splicing variant were no longer pursued.
Figure 2. FAIM increases NGF-induced neurite outgrowth. (A) Phase-contrast micrographs of PC12 cells stably transfected with empty vector (top), FAIM-S (middle), or FAIM-L (bottom). PC12 cells treated with complete medium (left) or with NGF 100 ng/ml (right) for (more ...)
To ascertain whether or not FAIM has a similar effect on NGF-induced neurite outgrowth in NGF-dependent primary neurons, we repeated these experiments using primary cultures of SCG neurons. Postnatal day (P1) mouse SCG neurons were plated and transiently cotransfected using gold carriers coated with both an eYFP expression plasmid (to visualize the transfected neurons) plus plasmids expressing FLAG-FAIM or the corresponding empty vector. The neurons were grown for 24 h in presence of NGF before the neuron arbors were captured in the confocal microscope and drawn using the confocal software. (A and B) illustrates drawings and pictures of representative SCG neurons showing an increase in the neuritic arbor in FAIM-transfected neurons compared with the cultures transfected with the empty vector. Quantification of neurite length and number of branching points shows that FAIM induced a significant increase in these parameters ( C). These results indicate that FAIM is capable of regulating neurite length in sympathetic neurons.
Figure 3. FAIM increases NGF-induced neurite outgrowth in mouse SCG neurons. Ballistic transfections of cultured neurons with an eYFP expression plasmid together with the Neo control vector or FAIM-S plasmids. After 24-h incubation with NGF (10 ng/ml), eYFP-labeled (more ...)
Endogenous FAIM affects NGF-induced neurite outgrowth
To ascertain the role of endogenous FAIM in NGF-induced neurite outgrowth, we generated two RNAi that target different sites of FAIM sequence. To check the ability of the RNAi constructs to knockdown FAIM expression, double stably transfected PC12 cell lines with FAIM plus the RNAi1, RNAi2, or the pSUPER empty vector were generated. Because anti-FAIM antibodies capable of detecting endogenous FAIM are not currently commercially available, we generated an anti-FAIM antibody (see Materials and methods). This antibody was only capable of recognizing overexpressed FAIM but was not sensitive enough to detect endogenous FAIM. Both RNAi1 and RNAi2 dramatically decreased the level of overexpressed FAIM protein in PC12 cells, RNAi2 being more effective than RNAi1 ( A). We also checked the level FAIM mRNA under these experimental conditions. As shown in B, transfection with the FAIM expression plasmid was associated with increased FAIM mRNA, whereas transfection with RNAi caused a decrease in endogenous FAIM mRNA.
Figure 4. FAIM-S is necessary for NGF-induced PC12 cells differentiation. (A) PC12 cells that stably express FAIM-S were transfected with pSUPER containing sequences encoding RNAi against FAIM (RNAi1 or RNAi2) or the empty vector (control). Stable pools of transfected (more ...)
To analyze the effect of RNAi on neurite length, PC12 cells were transfected with eYFP and either the FAIM RNAi1 and RNAi2 or the empty vector. After 4 d, the cells were treated with 100 ng/ml NGF plus 0.5% heat-inactivated horse serum for the indicated times after which neurite length was measured. C shows that both RNAi1 and RNAi2 decreased neurite outgrowth, the inhibition of outgrowth being significantly greater with RNAi2 than RNAi1. Similar results were obtained with PC12 cells stably transfected with the same constructs but in this case we observed a homogeneous effect on the entire population of transfected cells (). These decreases in neurite outgrowth with the RNAi constructs correlate with the level of FAIM protein knockdown and demonstrate that endogenous FAIM plays a role in regulating neurite outgrowth in NGF-stimulated PC12 cells.
To explore the relevance of endogenous FAIM in primary neurons, rat SCG neurons were transiently transfected with either RNAi2 or an empty vector (pSUPER). After 60 h of incubation with NGF, RNAi2 produced >30% significant decreases in total neurite length and branch point number compared with control-transfected neurons ( F). These results show that endogenous FAIM plays a significant role in NGF-induced neurite growth in sympathetic neurons.
ERK pathway is required for FAIM-induced neurite outgrowth
Because ERK signaling has been implicated in neurotrophin-induced neurite outgrowth, we investigated the role of this signaling pathway in mediating the neurite outgrowth–inducing effects of FAIM by using pharmacological agents to inhibit ERK signaling. Addition of PD98059, a MEK inhibitor, markedly reduced the extent of neurite outgrowth in FAIM-transfected cells ( A). These results suggest that activation of the ERK pathway is required for the neurite growth–inducing effect of FAIM overexpression. To confirm that the doses of PD98059 completely abolished ERK activation in FAIM-transfected cells as well as in the Neo cells, Western blot analysis was performed using a specific anti–phospho-ERK1/2 antibody. 30-min pretreatment of the stably transfected PC12 cells with 50 μM PD98059 completely abolished ERK1/2 phosphorylation ( B).
Figure 5. Effects of FAIM on ERK pathway stimulation. (A) PC12 cells stably transfected with empty vector or with FAIM-S were treated with NGF (100 ng/ml) or with NGF plus 50 μM of PD98059 for 1 d. Histogram shows the neurite length measurements of the (more ...)
Although the ERK pathway is required for the effects of FAIM overexpression on neurite outgrowth, to determine if FAIM stimulates ERK signaling, we used Western blotting to assess the level of ERK phosphorylation in NGF-stimulated PC12 cells stably transfected with FAIM, with the RNAi2 construct, or with an empty vector. NGF induced a peak of ERK phosphorylation after 5 min of stimulation. This increase in ERK1/2 phosphorylation was detectable after 15 min and 1 h and undetectable after 12 h. Our results show that the level of phosphorylation of ERK was not different between Neo, FAIM, or RNAi2 transfected cells at any of the time points studied ( C).
The absence of clear differences in the level and the duration of ERK activation between wild-type and FAIM-overexpressing or FAIM knockdown neurons raises the possibility that other signaling pathways play a role in mediating FAIM-induced neurite outgrowth.
FAIM enhances NGF-mediated activation of NF-κB
NGF signaling through p75NTR
and TrkA has been shown to be critical in the regulation of neurite outgrowth (Davies, 2000
), and one of the pathways activated by NGF through these receptors is the NF-κB pathway (Wood, 1995
; Carter et al., 1996
; Yoon et al., 1998
; Hamanoue et al., 1999
; Foehr et al., 2000
). To investigate whether or not FAIM-induced neurite outgrowth was mediated by NF-κB, FAIM stably transfected PC12 cells were transiently transfected with an NF-κB–dependent luciferase reporter construct or a control construct lacking the NF-κB site (Rodriguez et al., 1996
) and assayed for luciferase activity after NGF treatment. FAIM-transfected cells showed a marked increase in luciferase activity after 6–9 h of stimulation with NGF compared with the Neo-transfected cells, indicating that NF-κB activation is potentiated by FAIM expression ( A). In cells that were stably transfected with RNAi2, the levels of luciferase activity were found to be significantly decreased at all the time points analyzed ( A).
Figure 6. NF-κB activation is modulated by FAIM. (A) PC12 cells were stably transfected with an empty vector (Neo), with the FAIM-S expression plasmid, or with the FAIM RNAi plasmid. Cells were then transiently transfected with an NF-κB–dependent (more ...)
To determine an additional parameter of NF-κB activation, we checked p65 translocation to the nucleus after NGF stimulation. p65 is one of the two subunits most ubiquitous forms of NF-κB dimer. To this end, we stained cultures of FAIM-, RNAi2-, or Neo-transfected cells with an antibody against p65. Before NGF stimulation very few Neo-, FAIM-, or RNAi2-transfected cells showed nuclear staining for p65. After 5 min of NGF stimulation, cells stably transfected with FAIM or with the empty vector (Neo) have a similar activation of the NF-κB pathway as inferred from p65 nuclear translocation. Differences between Neo and FAIM are evident at longer stimulation. For example, at 15 min the percentage of Neo cells that show p65 translocation has dropped to ~15%, but remains high (~35%) in FAIM-transfected cells (). However, no increase was observed in RNAi2-transfected cells at any of the time points analyzed (). These results show that nuclear translocation of NF-κB following NGF stimulation is enhanced in cells overexpressing FAIM and completely prevented in cells in which FAIM expression was reduced by RNAi2. Together, these findings raised the possibility that NF-κB signaling plays a regulatory role in FAIM-induced neurite outgrowth and also suggest that endogenous FAIM is necessary for the NGF-induced NF-κB activation.
NF-κB signaling is required for FAIM-induced neurite outgrowth
Having ascertained that NF-κB activation is increased in FAIM-expressing PC12 cells stimulated with NGF, we investigated whether or not preventing NF-κB activation would interfere with FAIM neurite outgrowth responses. We stably (, A–D) or transiently ( E) transfected PC12 cells with a construct encoding a form of IκBα carrying serine-to-alanine mutations at residues 32 and 36, named SR-IκBα. These mutations prevent IκBα phosphorylation and subsequent proteasome-mediated degradation, thereby preventing release and nuclear translocation of NF-κB (Rodriguez et al., 1996
). Expression of SR-IκBα effectively prevented nuclear translocation of the p65 subunit of NF-κB even when the cells were treated with TNFα, one of the most potent activators of this pathway (). In these cells, NGF-induced neurite outgrowth was significantly reduced (Neo, 198 ± 18 μm, vs. SR-IκBα, 108 ± 5 μm; D) to a similar extent to that previously reported for NF-κB inhibition in PC12 cells (Foehr et al., 2000
), implying that NF-κB is involved in NGF-induced neurite outgrowth in PC12 cells.
Figure 7. SR-IκBα transfection attenuates the NGF-induced differentiation and abolishes the effects of FAIM overexpression on neurite outgrowth. (A) PC12 cells stably transfected with empty vector (Neo) or with SR-IκBα. Total cell (more ...)
We also analyzed whether or not this NF-κB is involved in the increased neurite outgrowth observed in FAIM-overexpressing PC12 cells after NGF stimulation. PC12 cells stably overexpressing FAIM-S were transiently transfected with the SR-IκBα together with eYFP. After 24 h, the cells were treated with NGF and neurite outgrowth was quantified 24 h later. SR-IκBα completely abolished FAIM-S–induced neurite outgrowth ( E). These results demonstrate that NF-κB signaling mediates the enhanced neurite outgrowth induced by FAIM overexpression.
p65-deficient cortical neurons show impaired FAIM-induced neurite outgrowth
To further confirm the role of NF-κB in mediating FAIM-induced neurite outgrowth, we compared the neurite arbors in cultures established from wild-type embryos and embryos with a null mutation of the p65
gene (Beg et al., 1995
). We used primary cultures of cortical neurons instead of SCG neurons for these studies because the latest stage at which these experiments could be performed was at day 15 of gestation (E15), since p65−/−
embryos die in utero shortly after this stage. After 3-h culture, E15 cortical neurons were ballistically transfected with gold particles coated with FAIM plus eYFP or pcDNA3 plus eYFP, and neurite arbors were analyzed after a further 24-h incubation. shows that p65+/+
cortical neurons overexpressing FAIM had more extensive (50–60% longer) and more branched neurite arbors (80–120% more branch points) than control transfected neurons. This increase in neurite growth and complexity between FAIM-transfected and control-transfected neurons was not observed in cultures established from p65−/−
littermates. These results additionally indicate that a functional NF-κB is required for FAIM-induced neurite outgrowth in cortical neurons.
Figure 8. FAIM-induced increase in neurite outgrowth is eliminated in cortical neurons from p65−/− mouse embryos. Cultured cortical neurons from p65+/+ (WT) or p65−/− (KO) mice were transfected with eYFP alone or with eYFP plus FAIM-S. (more ...)
FAIM interacts with TrkA and p75NTR receptors in an NGF-dependent manner
NF-κB activation after NGF stimulation has been described to be mediated by TrkA and p75NTR
(Carter et al., 1996
; Hamanoue et al., 1999
; Foehr et al., 2000
; Wooten et al., 2001
). To examine the possibility that FAIM interacts with the NGF receptors TrkA or p75, we performed immunoprecipitation experiments. shows that FLAG-tagged FAIM is able to coimmunoprecipitate with both HA-tagged TrkA ( A) and HA-tagged p75 ( B) receptors upon NGF stimulation when these molecules are transfected in PC12 cells. Lysates from cells that were not stimulated with NGF failed to show any interaction, indicating that this interaction requires NGF receptor activation. We also used immunoprecipitation to investigate whether FAIM is able to interact with endogenous TrkA and whether this interaction is dependent on ligand binding. PC12 cells were transfected with FLAG-tagged FAIM and stimulated with and without NGF. C shows that FLAG-tagged FAIM was able to coimmunoprecipitate with endogenous Trk but only when the cells had been stimulated with NGF. These data raise the possibility that FAIM influences NGF-induced neurite outgrowth by modulating activation of intracellular signaling pathways downstream of TrkA and p75.
Figure 9. FAIM interacts with TrkA and p75NTR receptors in an NGF-dependent manner. Wild-type PC12 cells were electroporated with FLAG-tagged FAIM-S and either TrkA-HA (A) or p75NTR-HA (B). After 48 h, the cells were stimulated (+) or not (−) with 100 ng/ml (more ...)