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The death receptor antagonist FAIM promotes neurite outgrowth by a mechanism that depends on ERK and NF-kapp B signaling.

Sole C, Dolcet X, Segura MF, Gutierrez H, Diaz-Meco MT, Gozzelino R, Sanchis D, Bayascas JR, Gallego C, Moscat J, Davies AM, Comella JX - J. Cell Biol. (2004)

Bottom Line: Whereas FAIM overexpression greatly enhanced neurite outgrowth from PC12 cells and sympathetic neurons grown with nerve growth factor (NGF), reduction of endogenous FAIM levels by RNAi decreased neurite outgrowth in these cells.The effect of FAIM on neurite outgrowth was also blocked by inhibition of the Ras-ERK pathway.These results reveal a new function of FAIM in promoting neurite outgrowth by a mechanism involving activation of the Ras-ERK pathway and NF-kappa B.

View Article: PubMed Central - PubMed

Affiliation: Group Cell Signalling and Apoptosis, Department of Basic Medical Sciences, Universitat de Lleida, 25008 Lleida, Spain.

ABSTRACT
Fas apoptosis inhibitory molecule (FAIM) is a protein identified as an antagonist of Fas-induced cell death. We show that FAIM overexpression fails to rescue neurons from trophic factor deprivation, but exerts a marked neurite growth-promoting action in different neuronal systems. Whereas FAIM overexpression greatly enhanced neurite outgrowth from PC12 cells and sympathetic neurons grown with nerve growth factor (NGF), reduction of endogenous FAIM levels by RNAi decreased neurite outgrowth in these cells. FAIM overexpression promoted NF-kappa B activation, and blocking this activation by using a super-repressor I kappa B alpha or by carrying out experiments using cortical neurons from mice that lack the p65 NF-kappa B subunit prevented FAIM-induced neurite outgrowth. The effect of FAIM on neurite outgrowth was also blocked by inhibition of the Ras-ERK pathway. Finally, we show that FAIM interacts with both Trk and p75 neurotrophin receptor NGF receptors in a ligand-dependent manner. These results reveal a new function of FAIM in promoting neurite outgrowth by a mechanism involving activation of the Ras-ERK pathway and NF-kappa B.

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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 cells were obtained with puromycin. Extracts were probed with the anti-FAIM antibody by Western blot analysis (top). Membranes were stripped and reprobed with an antibody to α-tubulin used to control the loading of lanes. (B) PC12 cells were stably transfected with an empty vector (control), with the FAIM-S expression plasmid, or with the plasmid RNAi1 or RNAi2. Total RNA (15 μg) was extracted, electrophoresed, and hybridized with a [32P]dCTP-labeled FAIM probe. Images were obtained by autoradiography for 40 h at −80°C. (C) PC12 cells were transiently transfected with eYFP and either RNAi1 or RNAi2, or with the empty vector (control). After 4 d, cells were treated with 100 ng/ml NGF for 1 d. Histogram shows the neurite length measurements of the labeled and digitally acquired cultures transfected with the indicated cDNA. (D and E) PC12 cells were stably transfected with either an empty vector (control) or with either of the two FAIM RNAi (RNAi1 and RNAi2). (D) Cells were treated with NGF for 1 or 3 d or left untreated and total neurite length was measured. (E) Representative images of NGF-induced differentiation on stably transfected cells after 1 d of treatment. Bar, 100 μm. (F) Rat SCG neurons were cotransfected with eYFP and either the empty pSUPER (control) or the vector containing RNAi2. After 60 h of NGF treatment (10 ng/ml), eYFP-labeled SCG were visualized, digitally acquired (representative images are shown on the left), and neurite outgrowth was quantified. Neurite length (top) and the branching points (bottom) were quantified. Bars, 25 μm.
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fig4: 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 cells were obtained with puromycin. Extracts were probed with the anti-FAIM antibody by Western blot analysis (top). Membranes were stripped and reprobed with an antibody to α-tubulin used to control the loading of lanes. (B) PC12 cells were stably transfected with an empty vector (control), with the FAIM-S expression plasmid, or with the plasmid RNAi1 or RNAi2. Total RNA (15 μg) was extracted, electrophoresed, and hybridized with a [32P]dCTP-labeled FAIM probe. Images were obtained by autoradiography for 40 h at −80°C. (C) PC12 cells were transiently transfected with eYFP and either RNAi1 or RNAi2, or with the empty vector (control). After 4 d, cells were treated with 100 ng/ml NGF for 1 d. Histogram shows the neurite length measurements of the labeled and digitally acquired cultures transfected with the indicated cDNA. (D and E) PC12 cells were stably transfected with either an empty vector (control) or with either of the two FAIM RNAi (RNAi1 and RNAi2). (D) Cells were treated with NGF for 1 or 3 d or left untreated and total neurite length was measured. (E) Representative images of NGF-induced differentiation on stably transfected cells after 1 d of treatment. Bar, 100 μm. (F) Rat SCG neurons were cotransfected with eYFP and either the empty pSUPER (control) or the vector containing RNAi2. After 60 h of NGF treatment (10 ng/ml), eYFP-labeled SCG were visualized, digitally acquired (representative images are shown on the left), and neurite outgrowth was quantified. Neurite length (top) and the branching points (bottom) were quantified. Bars, 25 μm.

Mentions: 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 (Fig. 4 A). We also checked the level FAIM mRNA under these experimental conditions. As shown in Fig. 4 B, transfection with the FAIM expression plasmid was associated with increased FAIM mRNA, whereas transfection with RNAi caused a decrease in endogenous FAIM mRNA.


The death receptor antagonist FAIM promotes neurite outgrowth by a mechanism that depends on ERK and NF-kapp B signaling.

Sole C, Dolcet X, Segura MF, Gutierrez H, Diaz-Meco MT, Gozzelino R, Sanchis D, Bayascas JR, Gallego C, Moscat J, Davies AM, Comella JX - J. Cell Biol. (2004)

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 cells were obtained with puromycin. Extracts were probed with the anti-FAIM antibody by Western blot analysis (top). Membranes were stripped and reprobed with an antibody to α-tubulin used to control the loading of lanes. (B) PC12 cells were stably transfected with an empty vector (control), with the FAIM-S expression plasmid, or with the plasmid RNAi1 or RNAi2. Total RNA (15 μg) was extracted, electrophoresed, and hybridized with a [32P]dCTP-labeled FAIM probe. Images were obtained by autoradiography for 40 h at −80°C. (C) PC12 cells were transiently transfected with eYFP and either RNAi1 or RNAi2, or with the empty vector (control). After 4 d, cells were treated with 100 ng/ml NGF for 1 d. Histogram shows the neurite length measurements of the labeled and digitally acquired cultures transfected with the indicated cDNA. (D and E) PC12 cells were stably transfected with either an empty vector (control) or with either of the two FAIM RNAi (RNAi1 and RNAi2). (D) Cells were treated with NGF for 1 or 3 d or left untreated and total neurite length was measured. (E) Representative images of NGF-induced differentiation on stably transfected cells after 1 d of treatment. Bar, 100 μm. (F) Rat SCG neurons were cotransfected with eYFP and either the empty pSUPER (control) or the vector containing RNAi2. After 60 h of NGF treatment (10 ng/ml), eYFP-labeled SCG were visualized, digitally acquired (representative images are shown on the left), and neurite outgrowth was quantified. Neurite length (top) and the branching points (bottom) were quantified. Bars, 25 μm.
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Related In: Results  -  Collection

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fig4: 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 cells were obtained with puromycin. Extracts were probed with the anti-FAIM antibody by Western blot analysis (top). Membranes were stripped and reprobed with an antibody to α-tubulin used to control the loading of lanes. (B) PC12 cells were stably transfected with an empty vector (control), with the FAIM-S expression plasmid, or with the plasmid RNAi1 or RNAi2. Total RNA (15 μg) was extracted, electrophoresed, and hybridized with a [32P]dCTP-labeled FAIM probe. Images were obtained by autoradiography for 40 h at −80°C. (C) PC12 cells were transiently transfected with eYFP and either RNAi1 or RNAi2, or with the empty vector (control). After 4 d, cells were treated with 100 ng/ml NGF for 1 d. Histogram shows the neurite length measurements of the labeled and digitally acquired cultures transfected with the indicated cDNA. (D and E) PC12 cells were stably transfected with either an empty vector (control) or with either of the two FAIM RNAi (RNAi1 and RNAi2). (D) Cells were treated with NGF for 1 or 3 d or left untreated and total neurite length was measured. (E) Representative images of NGF-induced differentiation on stably transfected cells after 1 d of treatment. Bar, 100 μm. (F) Rat SCG neurons were cotransfected with eYFP and either the empty pSUPER (control) or the vector containing RNAi2. After 60 h of NGF treatment (10 ng/ml), eYFP-labeled SCG were visualized, digitally acquired (representative images are shown on the left), and neurite outgrowth was quantified. Neurite length (top) and the branching points (bottom) were quantified. Bars, 25 μm.
Mentions: 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 (Fig. 4 A). We also checked the level FAIM mRNA under these experimental conditions. As shown in Fig. 4 B, transfection with the FAIM expression plasmid was associated with increased FAIM mRNA, whereas transfection with RNAi caused a decrease in endogenous FAIM mRNA.

Bottom Line: Whereas FAIM overexpression greatly enhanced neurite outgrowth from PC12 cells and sympathetic neurons grown with nerve growth factor (NGF), reduction of endogenous FAIM levels by RNAi decreased neurite outgrowth in these cells.The effect of FAIM on neurite outgrowth was also blocked by inhibition of the Ras-ERK pathway.These results reveal a new function of FAIM in promoting neurite outgrowth by a mechanism involving activation of the Ras-ERK pathway and NF-kappa B.

View Article: PubMed Central - PubMed

Affiliation: Group Cell Signalling and Apoptosis, Department of Basic Medical Sciences, Universitat de Lleida, 25008 Lleida, Spain.

ABSTRACT
Fas apoptosis inhibitory molecule (FAIM) is a protein identified as an antagonist of Fas-induced cell death. We show that FAIM overexpression fails to rescue neurons from trophic factor deprivation, but exerts a marked neurite growth-promoting action in different neuronal systems. Whereas FAIM overexpression greatly enhanced neurite outgrowth from PC12 cells and sympathetic neurons grown with nerve growth factor (NGF), reduction of endogenous FAIM levels by RNAi decreased neurite outgrowth in these cells. FAIM overexpression promoted NF-kappa B activation, and blocking this activation by using a super-repressor I kappa B alpha or by carrying out experiments using cortical neurons from mice that lack the p65 NF-kappa B subunit prevented FAIM-induced neurite outgrowth. The effect of FAIM on neurite outgrowth was also blocked by inhibition of the Ras-ERK pathway. Finally, we show that FAIM interacts with both Trk and p75 neurotrophin receptor NGF receptors in a ligand-dependent manner. These results reveal a new function of FAIM in promoting neurite outgrowth by a mechanism involving activation of the Ras-ERK pathway and NF-kappa B.

Show MeSH
Related in: MedlinePlus