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The PAAD/PYRIN-family protein ASC is a dual regulator of a conserved step in nuclear factor kappaB activation pathways.

Stehlik C, Fiorentino L, Dorfleutner A, Bruey JM, Ariza EM, Sagara J, Reed JC - J. Exp. Med. (2002)

Bottom Line: Apoptosis-associated speck-like protein containing a Caspase recruitment domain (ASC) belongs to a large family of proteins that contain a Pyrin, AIM, ASC, and death domain-like (PAAD) domain (also known as PYRIN, DAPIN, Pyk).Conversely, reducing endogenous levels of ASC using siRNA enhanced TNF- and LPS-induced degradation of the IKK substrate, IkappaBalpha.Our findings suggest that ASC modulates diverse NF-kappaB induction pathways by acting upon the IKK complex, implying a broad role for this and similar proteins containing PAAD domains in regulation of inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: The Burnham Institute, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Apoptosis-associated speck-like protein containing a Caspase recruitment domain (ASC) belongs to a large family of proteins that contain a Pyrin, AIM, ASC, and death domain-like (PAAD) domain (also known as PYRIN, DAPIN, Pyk). Recent data have suggested that ASC functions as an adaptor protein linking various PAAD-family proteins to pathways involved in nuclear factor (NF)-kappaB and pro-Caspase-1 activation. We present evidence here that the role of ASC in modulating NF-kappaB activation pathways is much broader than previously suspected, as it can either inhibit or activate NF-kappaB, depending on cellular context. While coexpression of ASC with certain PAAD-family proteins such as Pyrin and Cryopyrin increases NF-kappaB activity, ASC has an inhibitory influence on NF-kappaB activation by various proinflammatory stimuli, including tumor necrosis factor (TNF)alpha, interleukin 1beta, and lipopolysaccharide (LPS). Elevations in ASC protein levels or of the PAAD domain of ASC suppressed activation of IkappaB kinases in cells exposed to pro-inflammatory stimuli. Conversely, reducing endogenous levels of ASC using siRNA enhanced TNF- and LPS-induced degradation of the IKK substrate, IkappaBalpha. Our findings suggest that ASC modulates diverse NF-kappaB induction pathways by acting upon the IKK complex, implying a broad role for this and similar proteins containing PAAD domains in regulation of inflammatory responses.

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The PAAD of ASC regulates NF-κB activity, NF-κB DNA-binding activity, and expression of endogenous NF-κB target genes. (A–C) NF-κB activity as measured by reporter gene assays. Data represent fold induction (mean ± SD; n = 3) and are representative of several experiments. (A) NF-κB activity data are presented for HEK293N cells transfected with plasmids as indicated. (B) HEK293N cells were transfected with plasmids as indicated and NF-κB activity was measured after TNFα stimulation for 8 h. (C) Dose dependence of ASC-mediated inhibition of NF-κB activity, shown by transfecting increasing amounts of ASC-PAAD plasmid into HEK293N cells and measuring TNFα-induced NF-κB activity by reporter gene assay 1 d later. (D) NF-κB DNA-binding activity was measured by EMSA in nuclear lysates prepared from HEK293T cells that had been transiently transfected with the indicated plasmids. NIK(KK429,430AA) served as a control inhibiting TNF induction of NF-κB DNA-binding activity. As indicated, either IgG or anti-p65 antibodies were added for producing “super-shifted” DNA–protein complexes, or in lane 2 unlabeled NF-κB–binding DNA probe was included as a competitor for demonstrating binding specificity: band-shift (BS); super-shift (SS); free probe (FP). (E; top panel) Expression of ASC protein was measured by immunoblotting in THP-1 cells treated for the indicated times with 600 ng ml−1 LPS. Data represent quantification of scanned bands on blots using densitometry, normalized for Tubulin expression, and presented as a fold-increase relative to untreated cells. (Middle panel) HEK293N Myc-ASC-PAAD or Neo stable transfectants were assayed for NF-κB reporter gene activity at 8 h after stimulation with TNFα (mean ± SD; n = 3). (Bottom panel) Expression of Neo, ASC-PAAD in transient (trans.) and stable transfected HEK293N cells was compared with endogenous ASC in THP-1 cells untreated (−) or treated (+) for 6 h with LPS, by immunoblotting. Expression in the stable cell line was 1.5 times that of LPS stimulated and 2.9 times that of resting THP-1 cells. (F) HEK293N-Neo and Myc-ASC-PAAD stable transfectants were transiently transfected with plasmids encoding GFP or GFP-antisense ASC together with a NF-κB-luciferase reporter plasmid and NF-κB activity was measured 8 h after TNFα stimulation. Alternatively, lysates were analyzed by immunoblotting with anti-GFP and anti-Myc antibodies. Note that the shift in molecular weight of GFP in antisense ASC-transfected cells is due to an artificial open reading frame, created by insertion of the antisense cDNA into pEGFP. (G) HEK293N cells were transiently transfected with either Neo control or Myc-ASC-PAAD plasmids, treated with TNFα for 4 h where indicated and lysates were SDS-PAGE/immunoblotted using anti-TRAF1, anti-TRAF2, and anti-Tubulin antibodies to measure expression of the endogenous proteins. (H) Alternatively, total RNA was isolated from stably transfected HEK293N cells and analyzed by RT-PCR for TRAF1 and GAPDH. Lanes represent: HEK293N-Neo cells (1) untreated and (2) treated for 4 h with TNFα; and HEK293N-ASC-PAAD cells (3) untreated and (4) treated for 4 h with TNFα; (M) molecular size marker. (I) THP-1 cells that had been stably transfected with plasmids encoding GFP or GFP-ASC-PAAD were treated with LPS as indicated and lysates were subject to SDS-PAGE/immunoblotting using anti- ICAM-1, anti-GFP, and anti-βActin antibodies.
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fig2: The PAAD of ASC regulates NF-κB activity, NF-κB DNA-binding activity, and expression of endogenous NF-κB target genes. (A–C) NF-κB activity as measured by reporter gene assays. Data represent fold induction (mean ± SD; n = 3) and are representative of several experiments. (A) NF-κB activity data are presented for HEK293N cells transfected with plasmids as indicated. (B) HEK293N cells were transfected with plasmids as indicated and NF-κB activity was measured after TNFα stimulation for 8 h. (C) Dose dependence of ASC-mediated inhibition of NF-κB activity, shown by transfecting increasing amounts of ASC-PAAD plasmid into HEK293N cells and measuring TNFα-induced NF-κB activity by reporter gene assay 1 d later. (D) NF-κB DNA-binding activity was measured by EMSA in nuclear lysates prepared from HEK293T cells that had been transiently transfected with the indicated plasmids. NIK(KK429,430AA) served as a control inhibiting TNF induction of NF-κB DNA-binding activity. As indicated, either IgG or anti-p65 antibodies were added for producing “super-shifted” DNA–protein complexes, or in lane 2 unlabeled NF-κB–binding DNA probe was included as a competitor for demonstrating binding specificity: band-shift (BS); super-shift (SS); free probe (FP). (E; top panel) Expression of ASC protein was measured by immunoblotting in THP-1 cells treated for the indicated times with 600 ng ml−1 LPS. Data represent quantification of scanned bands on blots using densitometry, normalized for Tubulin expression, and presented as a fold-increase relative to untreated cells. (Middle panel) HEK293N Myc-ASC-PAAD or Neo stable transfectants were assayed for NF-κB reporter gene activity at 8 h after stimulation with TNFα (mean ± SD; n = 3). (Bottom panel) Expression of Neo, ASC-PAAD in transient (trans.) and stable transfected HEK293N cells was compared with endogenous ASC in THP-1 cells untreated (−) or treated (+) for 6 h with LPS, by immunoblotting. Expression in the stable cell line was 1.5 times that of LPS stimulated and 2.9 times that of resting THP-1 cells. (F) HEK293N-Neo and Myc-ASC-PAAD stable transfectants were transiently transfected with plasmids encoding GFP or GFP-antisense ASC together with a NF-κB-luciferase reporter plasmid and NF-κB activity was measured 8 h after TNFα stimulation. Alternatively, lysates were analyzed by immunoblotting with anti-GFP and anti-Myc antibodies. Note that the shift in molecular weight of GFP in antisense ASC-transfected cells is due to an artificial open reading frame, created by insertion of the antisense cDNA into pEGFP. (G) HEK293N cells were transiently transfected with either Neo control or Myc-ASC-PAAD plasmids, treated with TNFα for 4 h where indicated and lysates were SDS-PAGE/immunoblotted using anti-TRAF1, anti-TRAF2, and anti-Tubulin antibodies to measure expression of the endogenous proteins. (H) Alternatively, total RNA was isolated from stably transfected HEK293N cells and analyzed by RT-PCR for TRAF1 and GAPDH. Lanes represent: HEK293N-Neo cells (1) untreated and (2) treated for 4 h with TNFα; and HEK293N-ASC-PAAD cells (3) untreated and (4) treated for 4 h with TNFα; (M) molecular size marker. (I) THP-1 cells that had been stably transfected with plasmids encoding GFP or GFP-ASC-PAAD were treated with LPS as indicated and lysates were subject to SDS-PAGE/immunoblotting using anti- ICAM-1, anti-GFP, and anti-βActin antibodies.

Mentions: To map the domain in ASC responsible for modulation of NF-κB activity, we compared the effects of full-length ASC to truncation mutants containing only the PAAD or CARD domains. Neither the PAAD nor the CARD of ASC induced NF-κB activity when expressed by transient transfection in cell lines such as HEK293N (Fig. 2 A), consistent with a previous report (24). In TNFα-stimulated cells, both full-length ASC and the PAAD of ASC profoundly suppressed NF-κB activity, while the CARD of ASC did not (Fig. 2 B). Inhibition of TNFα-induced NF-κB activity by the PAAD of ASC was dose-dependent (Fig. 2 C). NF-κB DNA binding activity was also reduced by overexpression of the PAAD of ASC, as measured by EMSAs using a DNA probe containing NF-κB binding sites (Fig. 2 D), and correlated with reduced translocation of the p65 subunit of NF-κB into the nuclei of TNFα-stimulated cells, as visualized by immunofluorescence microscopy analysis (unpublished data).


The PAAD/PYRIN-family protein ASC is a dual regulator of a conserved step in nuclear factor kappaB activation pathways.

Stehlik C, Fiorentino L, Dorfleutner A, Bruey JM, Ariza EM, Sagara J, Reed JC - J. Exp. Med. (2002)

The PAAD of ASC regulates NF-κB activity, NF-κB DNA-binding activity, and expression of endogenous NF-κB target genes. (A–C) NF-κB activity as measured by reporter gene assays. Data represent fold induction (mean ± SD; n = 3) and are representative of several experiments. (A) NF-κB activity data are presented for HEK293N cells transfected with plasmids as indicated. (B) HEK293N cells were transfected with plasmids as indicated and NF-κB activity was measured after TNFα stimulation for 8 h. (C) Dose dependence of ASC-mediated inhibition of NF-κB activity, shown by transfecting increasing amounts of ASC-PAAD plasmid into HEK293N cells and measuring TNFα-induced NF-κB activity by reporter gene assay 1 d later. (D) NF-κB DNA-binding activity was measured by EMSA in nuclear lysates prepared from HEK293T cells that had been transiently transfected with the indicated plasmids. NIK(KK429,430AA) served as a control inhibiting TNF induction of NF-κB DNA-binding activity. As indicated, either IgG or anti-p65 antibodies were added for producing “super-shifted” DNA–protein complexes, or in lane 2 unlabeled NF-κB–binding DNA probe was included as a competitor for demonstrating binding specificity: band-shift (BS); super-shift (SS); free probe (FP). (E; top panel) Expression of ASC protein was measured by immunoblotting in THP-1 cells treated for the indicated times with 600 ng ml−1 LPS. Data represent quantification of scanned bands on blots using densitometry, normalized for Tubulin expression, and presented as a fold-increase relative to untreated cells. (Middle panel) HEK293N Myc-ASC-PAAD or Neo stable transfectants were assayed for NF-κB reporter gene activity at 8 h after stimulation with TNFα (mean ± SD; n = 3). (Bottom panel) Expression of Neo, ASC-PAAD in transient (trans.) and stable transfected HEK293N cells was compared with endogenous ASC in THP-1 cells untreated (−) or treated (+) for 6 h with LPS, by immunoblotting. Expression in the stable cell line was 1.5 times that of LPS stimulated and 2.9 times that of resting THP-1 cells. (F) HEK293N-Neo and Myc-ASC-PAAD stable transfectants were transiently transfected with plasmids encoding GFP or GFP-antisense ASC together with a NF-κB-luciferase reporter plasmid and NF-κB activity was measured 8 h after TNFα stimulation. Alternatively, lysates were analyzed by immunoblotting with anti-GFP and anti-Myc antibodies. Note that the shift in molecular weight of GFP in antisense ASC-transfected cells is due to an artificial open reading frame, created by insertion of the antisense cDNA into pEGFP. (G) HEK293N cells were transiently transfected with either Neo control or Myc-ASC-PAAD plasmids, treated with TNFα for 4 h where indicated and lysates were SDS-PAGE/immunoblotted using anti-TRAF1, anti-TRAF2, and anti-Tubulin antibodies to measure expression of the endogenous proteins. (H) Alternatively, total RNA was isolated from stably transfected HEK293N cells and analyzed by RT-PCR for TRAF1 and GAPDH. Lanes represent: HEK293N-Neo cells (1) untreated and (2) treated for 4 h with TNFα; and HEK293N-ASC-PAAD cells (3) untreated and (4) treated for 4 h with TNFα; (M) molecular size marker. (I) THP-1 cells that had been stably transfected with plasmids encoding GFP or GFP-ASC-PAAD were treated with LPS as indicated and lysates were subject to SDS-PAGE/immunoblotting using anti- ICAM-1, anti-GFP, and anti-βActin antibodies.
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fig2: The PAAD of ASC regulates NF-κB activity, NF-κB DNA-binding activity, and expression of endogenous NF-κB target genes. (A–C) NF-κB activity as measured by reporter gene assays. Data represent fold induction (mean ± SD; n = 3) and are representative of several experiments. (A) NF-κB activity data are presented for HEK293N cells transfected with plasmids as indicated. (B) HEK293N cells were transfected with plasmids as indicated and NF-κB activity was measured after TNFα stimulation for 8 h. (C) Dose dependence of ASC-mediated inhibition of NF-κB activity, shown by transfecting increasing amounts of ASC-PAAD plasmid into HEK293N cells and measuring TNFα-induced NF-κB activity by reporter gene assay 1 d later. (D) NF-κB DNA-binding activity was measured by EMSA in nuclear lysates prepared from HEK293T cells that had been transiently transfected with the indicated plasmids. NIK(KK429,430AA) served as a control inhibiting TNF induction of NF-κB DNA-binding activity. As indicated, either IgG or anti-p65 antibodies were added for producing “super-shifted” DNA–protein complexes, or in lane 2 unlabeled NF-κB–binding DNA probe was included as a competitor for demonstrating binding specificity: band-shift (BS); super-shift (SS); free probe (FP). (E; top panel) Expression of ASC protein was measured by immunoblotting in THP-1 cells treated for the indicated times with 600 ng ml−1 LPS. Data represent quantification of scanned bands on blots using densitometry, normalized for Tubulin expression, and presented as a fold-increase relative to untreated cells. (Middle panel) HEK293N Myc-ASC-PAAD or Neo stable transfectants were assayed for NF-κB reporter gene activity at 8 h after stimulation with TNFα (mean ± SD; n = 3). (Bottom panel) Expression of Neo, ASC-PAAD in transient (trans.) and stable transfected HEK293N cells was compared with endogenous ASC in THP-1 cells untreated (−) or treated (+) for 6 h with LPS, by immunoblotting. Expression in the stable cell line was 1.5 times that of LPS stimulated and 2.9 times that of resting THP-1 cells. (F) HEK293N-Neo and Myc-ASC-PAAD stable transfectants were transiently transfected with plasmids encoding GFP or GFP-antisense ASC together with a NF-κB-luciferase reporter plasmid and NF-κB activity was measured 8 h after TNFα stimulation. Alternatively, lysates were analyzed by immunoblotting with anti-GFP and anti-Myc antibodies. Note that the shift in molecular weight of GFP in antisense ASC-transfected cells is due to an artificial open reading frame, created by insertion of the antisense cDNA into pEGFP. (G) HEK293N cells were transiently transfected with either Neo control or Myc-ASC-PAAD plasmids, treated with TNFα for 4 h where indicated and lysates were SDS-PAGE/immunoblotted using anti-TRAF1, anti-TRAF2, and anti-Tubulin antibodies to measure expression of the endogenous proteins. (H) Alternatively, total RNA was isolated from stably transfected HEK293N cells and analyzed by RT-PCR for TRAF1 and GAPDH. Lanes represent: HEK293N-Neo cells (1) untreated and (2) treated for 4 h with TNFα; and HEK293N-ASC-PAAD cells (3) untreated and (4) treated for 4 h with TNFα; (M) molecular size marker. (I) THP-1 cells that had been stably transfected with plasmids encoding GFP or GFP-ASC-PAAD were treated with LPS as indicated and lysates were subject to SDS-PAGE/immunoblotting using anti- ICAM-1, anti-GFP, and anti-βActin antibodies.
Mentions: To map the domain in ASC responsible for modulation of NF-κB activity, we compared the effects of full-length ASC to truncation mutants containing only the PAAD or CARD domains. Neither the PAAD nor the CARD of ASC induced NF-κB activity when expressed by transient transfection in cell lines such as HEK293N (Fig. 2 A), consistent with a previous report (24). In TNFα-stimulated cells, both full-length ASC and the PAAD of ASC profoundly suppressed NF-κB activity, while the CARD of ASC did not (Fig. 2 B). Inhibition of TNFα-induced NF-κB activity by the PAAD of ASC was dose-dependent (Fig. 2 C). NF-κB DNA binding activity was also reduced by overexpression of the PAAD of ASC, as measured by EMSAs using a DNA probe containing NF-κB binding sites (Fig. 2 D), and correlated with reduced translocation of the p65 subunit of NF-κB into the nuclei of TNFα-stimulated cells, as visualized by immunofluorescence microscopy analysis (unpublished data).

Bottom Line: Apoptosis-associated speck-like protein containing a Caspase recruitment domain (ASC) belongs to a large family of proteins that contain a Pyrin, AIM, ASC, and death domain-like (PAAD) domain (also known as PYRIN, DAPIN, Pyk).Conversely, reducing endogenous levels of ASC using siRNA enhanced TNF- and LPS-induced degradation of the IKK substrate, IkappaBalpha.Our findings suggest that ASC modulates diverse NF-kappaB induction pathways by acting upon the IKK complex, implying a broad role for this and similar proteins containing PAAD domains in regulation of inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: The Burnham Institute, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Apoptosis-associated speck-like protein containing a Caspase recruitment domain (ASC) belongs to a large family of proteins that contain a Pyrin, AIM, ASC, and death domain-like (PAAD) domain (also known as PYRIN, DAPIN, Pyk). Recent data have suggested that ASC functions as an adaptor protein linking various PAAD-family proteins to pathways involved in nuclear factor (NF)-kappaB and pro-Caspase-1 activation. We present evidence here that the role of ASC in modulating NF-kappaB activation pathways is much broader than previously suspected, as it can either inhibit or activate NF-kappaB, depending on cellular context. While coexpression of ASC with certain PAAD-family proteins such as Pyrin and Cryopyrin increases NF-kappaB activity, ASC has an inhibitory influence on NF-kappaB activation by various proinflammatory stimuli, including tumor necrosis factor (TNF)alpha, interleukin 1beta, and lipopolysaccharide (LPS). Elevations in ASC protein levels or of the PAAD domain of ASC suppressed activation of IkappaB kinases in cells exposed to pro-inflammatory stimuli. Conversely, reducing endogenous levels of ASC using siRNA enhanced TNF- and LPS-induced degradation of the IKK substrate, IkappaBalpha. Our findings suggest that ASC modulates diverse NF-kappaB induction pathways by acting upon the IKK complex, implying a broad role for this and similar proteins containing PAAD domains in regulation of inflammatory responses.

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Related in: MedlinePlus