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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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Differential effects of ASC on NF-κB activation. (A and B) HEK293T cells were transfected with either 200 ng of control plasmid or 200 ng of various plasmids encoding PAAD-family members (ASC, NAC, PAN1, PAN2, Pyrin, or Cryopyrin) alone (gray bars) or in combination with 200 ng of ASC-encoding plasmid (black bars). Transfections also included 100 ng of pNF-κB and 6 ng of pRL-TK, maintaining the total DNA of transfections at 1 μg. At 36 h after transfection, cells were either left untreated (A) or stimulated with TNFα for 8 h (B) and analyzed for NF-κB activity by reporter gene assay. Data represent the mean ± SD (n = 3) and are expressed as fold-induction relative to cells transfected with pcDNA3 control plasmid without TNFα stimulation, and are representative of several experiments. (C) HEK293N cells, transfected as above, were cotransfected with IL-1RI and IL-1RAcP and induced with IL-1β for 8 h. (D and E) Cells were transfected with plasmids encoding Bcl-10 (D) or Nod (E) alone or in combination with ASC, and NF-κB activity was measured. (F and G) To assess specificity of NF-κB reporter gene assays in HEK293N cells, p53 transcriptional activity was determined after transfection with p53-responsive luciferase reporter plasmid pRL-p53 and stimulation the next day with DNA-damaging drug doxorubicin (DOX) (40 ng ml−1) (F), or AP-1 transcriptional activity was determined after transfection with control or TRAF2-encoding plasmids with or without ASC and the AP-1–responsive plasmid pRL-AP1 (G). Transcriptional activity was measured 1 d later by reporter gene assay (fold induction, mean ± SD; n = 3).
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fig1: Differential effects of ASC on NF-κB activation. (A and B) HEK293T cells were transfected with either 200 ng of control plasmid or 200 ng of various plasmids encoding PAAD-family members (ASC, NAC, PAN1, PAN2, Pyrin, or Cryopyrin) alone (gray bars) or in combination with 200 ng of ASC-encoding plasmid (black bars). Transfections also included 100 ng of pNF-κB and 6 ng of pRL-TK, maintaining the total DNA of transfections at 1 μg. At 36 h after transfection, cells were either left untreated (A) or stimulated with TNFα for 8 h (B) and analyzed for NF-κB activity by reporter gene assay. Data represent the mean ± SD (n = 3) and are expressed as fold-induction relative to cells transfected with pcDNA3 control plasmid without TNFα stimulation, and are representative of several experiments. (C) HEK293N cells, transfected as above, were cotransfected with IL-1RI and IL-1RAcP and induced with IL-1β for 8 h. (D and E) Cells were transfected with plasmids encoding Bcl-10 (D) or Nod (E) alone or in combination with ASC, and NF-κB activity was measured. (F and G) To assess specificity of NF-κB reporter gene assays in HEK293N cells, p53 transcriptional activity was determined after transfection with p53-responsive luciferase reporter plasmid pRL-p53 and stimulation the next day with DNA-damaging drug doxorubicin (DOX) (40 ng ml−1) (F), or AP-1 transcriptional activity was determined after transfection with control or TRAF2-encoding plasmids with or without ASC and the AP-1–responsive plasmid pRL-AP1 (G). Transcriptional activity was measured 1 d later by reporter gene assay (fold induction, mean ± SD; n = 3).

Mentions: Recently, it was reported that coexpression of ASC with Cryopyrin (PYPAF-1/NALP3) or PYPAF-7 (PAN6) induces NF-κB activity in transient transfection reporter gene assays performed in HEK293T cells (20, 21). These cells contain essentially no detectable ASC (unpublished data), thus avoiding contributions of the endogenous ASC protein. Similar to previous reports, we observed 20–70-fold inductions in NF-κB activity, when ASC was coexpressed with Cryopyrin or Pyrin in HEK293T cells, as measured by reporter gene assays in HEK293T cells (Fig. 1 A). In contrast, neither ASC, nor Pyrin or Cryopyrin alone induced significant NF-κB activity. The ability of ASC to collaborate with other PAAD-containing proteins in NF-κB induction was selective, as coexpression with NAC (NALP1, DEFCAP, CARD7), PAN1 (PYPAF-2, NALP2, NBS1), or PAN2 (PYPAF-4, NALP4) did not result in significant NF-κB activity.


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)

Differential effects of ASC on NF-κB activation. (A and B) HEK293T cells were transfected with either 200 ng of control plasmid or 200 ng of various plasmids encoding PAAD-family members (ASC, NAC, PAN1, PAN2, Pyrin, or Cryopyrin) alone (gray bars) or in combination with 200 ng of ASC-encoding plasmid (black bars). Transfections also included 100 ng of pNF-κB and 6 ng of pRL-TK, maintaining the total DNA of transfections at 1 μg. At 36 h after transfection, cells were either left untreated (A) or stimulated with TNFα for 8 h (B) and analyzed for NF-κB activity by reporter gene assay. Data represent the mean ± SD (n = 3) and are expressed as fold-induction relative to cells transfected with pcDNA3 control plasmid without TNFα stimulation, and are representative of several experiments. (C) HEK293N cells, transfected as above, were cotransfected with IL-1RI and IL-1RAcP and induced with IL-1β for 8 h. (D and E) Cells were transfected with plasmids encoding Bcl-10 (D) or Nod (E) alone or in combination with ASC, and NF-κB activity was measured. (F and G) To assess specificity of NF-κB reporter gene assays in HEK293N cells, p53 transcriptional activity was determined after transfection with p53-responsive luciferase reporter plasmid pRL-p53 and stimulation the next day with DNA-damaging drug doxorubicin (DOX) (40 ng ml−1) (F), or AP-1 transcriptional activity was determined after transfection with control or TRAF2-encoding plasmids with or without ASC and the AP-1–responsive plasmid pRL-AP1 (G). Transcriptional activity was measured 1 d later by reporter gene assay (fold induction, mean ± SD; n = 3).
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fig1: Differential effects of ASC on NF-κB activation. (A and B) HEK293T cells were transfected with either 200 ng of control plasmid or 200 ng of various plasmids encoding PAAD-family members (ASC, NAC, PAN1, PAN2, Pyrin, or Cryopyrin) alone (gray bars) or in combination with 200 ng of ASC-encoding plasmid (black bars). Transfections also included 100 ng of pNF-κB and 6 ng of pRL-TK, maintaining the total DNA of transfections at 1 μg. At 36 h after transfection, cells were either left untreated (A) or stimulated with TNFα for 8 h (B) and analyzed for NF-κB activity by reporter gene assay. Data represent the mean ± SD (n = 3) and are expressed as fold-induction relative to cells transfected with pcDNA3 control plasmid without TNFα stimulation, and are representative of several experiments. (C) HEK293N cells, transfected as above, were cotransfected with IL-1RI and IL-1RAcP and induced with IL-1β for 8 h. (D and E) Cells were transfected with plasmids encoding Bcl-10 (D) or Nod (E) alone or in combination with ASC, and NF-κB activity was measured. (F and G) To assess specificity of NF-κB reporter gene assays in HEK293N cells, p53 transcriptional activity was determined after transfection with p53-responsive luciferase reporter plasmid pRL-p53 and stimulation the next day with DNA-damaging drug doxorubicin (DOX) (40 ng ml−1) (F), or AP-1 transcriptional activity was determined after transfection with control or TRAF2-encoding plasmids with or without ASC and the AP-1–responsive plasmid pRL-AP1 (G). Transcriptional activity was measured 1 d later by reporter gene assay (fold induction, mean ± SD; n = 3).
Mentions: Recently, it was reported that coexpression of ASC with Cryopyrin (PYPAF-1/NALP3) or PYPAF-7 (PAN6) induces NF-κB activity in transient transfection reporter gene assays performed in HEK293T cells (20, 21). These cells contain essentially no detectable ASC (unpublished data), thus avoiding contributions of the endogenous ASC protein. Similar to previous reports, we observed 20–70-fold inductions in NF-κB activity, when ASC was coexpressed with Cryopyrin or Pyrin in HEK293T cells, as measured by reporter gene assays in HEK293T cells (Fig. 1 A). In contrast, neither ASC, nor Pyrin or Cryopyrin alone induced significant NF-κB activity. The ability of ASC to collaborate with other PAAD-containing proteins in NF-κB induction was selective, as coexpression with NAC (NALP1, DEFCAP, CARD7), PAN1 (PYPAF-2, NALP2, NBS1), or PAN2 (PYPAF-4, NALP4) did not result in significant NF-κB activity.

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.

Show MeSH
Related in: MedlinePlus