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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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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 lysates were analyzed by Western blot using an antibody against IκBα (top). Membranes were stripped and reprobed with an antibody to α-tubulin to normalize the loading content per lane (bottom). (B–D) PC12 cells stably transfected with the empty vector (Neo) or with the vector expressing SR-IκBα and were treated with NGF for 5 min, TNFα for 15 min, or left untreated. Quantification of immunocytochemical detection of nuclear p65 is shown in B. Representative pictures of low and high magnification (arrows point to selected cells shown in insets) are shown in C. Bar, 25 μm. Quantification of neurite outgrowth was performed 1 d after NGF treatment (D). Asterisks indicate a significant (P < 0.001; t test) reduction of neurite outgrowth in PC12 cells expressing the SR-IκBα. (E) Neo or FAIM-S stably transfected cells were additionally transfected transiently with eYFP alone or with eYFP plus SR-IκBα. After 24 h, they were treated with NGF (100 ng/ml) for an additional 24-h period and neurite length was quantified. Significant differences are indicated (*, P < 0.001; t test).
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fig7: 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 lysates were analyzed by Western blot using an antibody against IκBα (top). Membranes were stripped and reprobed with an antibody to α-tubulin to normalize the loading content per lane (bottom). (B–D) PC12 cells stably transfected with the empty vector (Neo) or with the vector expressing SR-IκBα and were treated with NGF for 5 min, TNFα for 15 min, or left untreated. Quantification of immunocytochemical detection of nuclear p65 is shown in B. Representative pictures of low and high magnification (arrows point to selected cells shown in insets) are shown in C. Bar, 25 μm. Quantification of neurite outgrowth was performed 1 d after NGF treatment (D). Asterisks indicate a significant (P < 0.001; t test) reduction of neurite outgrowth in PC12 cells expressing the SR-IκBα. (E) Neo or FAIM-S stably transfected cells were additionally transfected transiently with eYFP alone or with eYFP plus SR-IκBα. After 24 h, they were treated with NGF (100 ng/ml) for an additional 24-h period and neurite length was quantified. Significant differences are indicated (*, P < 0.001; t test).

Mentions: 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 (Fig. 7, A–D) or transiently (Fig. 7 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 (Fig. 7, B and C). In these cells, NGF-induced neurite outgrowth was significantly reduced (Neo, 198 ± 18 μm, vs. SR-IκBα, 108 ± 5 μm; Fig. 7 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.


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)

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 lysates were analyzed by Western blot using an antibody against IκBα (top). Membranes were stripped and reprobed with an antibody to α-tubulin to normalize the loading content per lane (bottom). (B–D) PC12 cells stably transfected with the empty vector (Neo) or with the vector expressing SR-IκBα and were treated with NGF for 5 min, TNFα for 15 min, or left untreated. Quantification of immunocytochemical detection of nuclear p65 is shown in B. Representative pictures of low and high magnification (arrows point to selected cells shown in insets) are shown in C. Bar, 25 μm. Quantification of neurite outgrowth was performed 1 d after NGF treatment (D). Asterisks indicate a significant (P < 0.001; t test) reduction of neurite outgrowth in PC12 cells expressing the SR-IκBα. (E) Neo or FAIM-S stably transfected cells were additionally transfected transiently with eYFP alone or with eYFP plus SR-IκBα. After 24 h, they were treated with NGF (100 ng/ml) for an additional 24-h period and neurite length was quantified. Significant differences are indicated (*, P < 0.001; t test).
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fig7: 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 lysates were analyzed by Western blot using an antibody against IκBα (top). Membranes were stripped and reprobed with an antibody to α-tubulin to normalize the loading content per lane (bottom). (B–D) PC12 cells stably transfected with the empty vector (Neo) or with the vector expressing SR-IκBα and were treated with NGF for 5 min, TNFα for 15 min, or left untreated. Quantification of immunocytochemical detection of nuclear p65 is shown in B. Representative pictures of low and high magnification (arrows point to selected cells shown in insets) are shown in C. Bar, 25 μm. Quantification of neurite outgrowth was performed 1 d after NGF treatment (D). Asterisks indicate a significant (P < 0.001; t test) reduction of neurite outgrowth in PC12 cells expressing the SR-IκBα. (E) Neo or FAIM-S stably transfected cells were additionally transfected transiently with eYFP alone or with eYFP plus SR-IκBα. After 24 h, they were treated with NGF (100 ng/ml) for an additional 24-h period and neurite length was quantified. Significant differences are indicated (*, P < 0.001; t test).
Mentions: 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 (Fig. 7, A–D) or transiently (Fig. 7 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 (Fig. 7, B and C). In these cells, NGF-induced neurite outgrowth was significantly reduced (Neo, 198 ± 18 μm, vs. SR-IκBα, 108 ± 5 μm; Fig. 7 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.

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