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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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ASC regulates IκBα levels. (A) Endogenous IκBα expression was analyzed by immunoblotting in stably transfected HEK293N-Neo or ASC-PAAD cells at various times following stimulation with TNFα. The position of unphosphorylated and phosphorylated IκBα proteins are indicated. (B) MCF7 cells were transfected on two consecutive days with two different double-strand ASC siRNAs (ASC nucleotides 721–741 [+1]; and nucleotides 474–494 [+2]) or control (c) siRNA using Oligofectamine. After 24 h, cells were either left untreated (−) or treated with 10 ng ml−1 TNFα for 10 min (+). Cell lysates were prepared, normalized for protein content, and analyzed by immunoblotting with antibodies specific for ASC (top), IκBα (middle), or βActin (bottom). (C) TPA-differentiated THP-1 cells were transfected with ASC (ASC nucleotides 474–494 [+2]) or control (c) siRNA on two consecutive days, then stimulated the following day for 30 min with LPS. Cell lysates were analyzed by immunoblotting for ASC (top), IκBα (middle), or βActin (bottom). (B and C) Blots were also quantified by scanning-densitometry analysis of the autoradiograms, and are presented below the gel as a bar graph showing fold expression compared with uninduced cells, normalized to βActin expression as determined by immunoblotting.
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fig5: ASC regulates IκBα levels. (A) Endogenous IκBα expression was analyzed by immunoblotting in stably transfected HEK293N-Neo or ASC-PAAD cells at various times following stimulation with TNFα. The position of unphosphorylated and phosphorylated IκBα proteins are indicated. (B) MCF7 cells were transfected on two consecutive days with two different double-strand ASC siRNAs (ASC nucleotides 721–741 [+1]; and nucleotides 474–494 [+2]) or control (c) siRNA using Oligofectamine. After 24 h, cells were either left untreated (−) or treated with 10 ng ml−1 TNFα for 10 min (+). Cell lysates were prepared, normalized for protein content, and analyzed by immunoblotting with antibodies specific for ASC (top), IκBα (middle), or βActin (bottom). (C) TPA-differentiated THP-1 cells were transfected with ASC (ASC nucleotides 474–494 [+2]) or control (c) siRNA on two consecutive days, then stimulated the following day for 30 min with LPS. Cell lysates were analyzed by immunoblotting for ASC (top), IκBα (middle), or βActin (bottom). (B and C) Blots were also quantified by scanning-densitometry analysis of the autoradiograms, and are presented below the gel as a bar graph showing fold expression compared with uninduced cells, normalized to βActin expression as determined by immunoblotting.

Mentions: As IKK activity induces phosphorylation and subsequent degradation of IκB-family proteins, we evaluated the effects of the PAAD of ASC on levels of endogenous IκBα in these stably transfected cells, before and at various times after TNFα stimulation. Immunoblot analysis of lysates from HEK293N-Neo cells using anti-IκBα antibody demonstrated the appearance of a doublet band (indicative of phosphorylation of IκBα) within 5 min after TNFα simulation, followed by disappearance of IκBα protein. In contrast, IκBα protein levels were sustained at detectable levels in ASC-PAAD–expressing HEK293N cells despite TNFα treatment. Furthermore, the IκBα doublet band indicative of phosphorylation was not observed until much later, at 15–30 min after stimulation (Fig. 5 A), thus demonstrating a marked delay relative to control cells.


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)

ASC regulates IκBα levels. (A) Endogenous IκBα expression was analyzed by immunoblotting in stably transfected HEK293N-Neo or ASC-PAAD cells at various times following stimulation with TNFα. The position of unphosphorylated and phosphorylated IκBα proteins are indicated. (B) MCF7 cells were transfected on two consecutive days with two different double-strand ASC siRNAs (ASC nucleotides 721–741 [+1]; and nucleotides 474–494 [+2]) or control (c) siRNA using Oligofectamine. After 24 h, cells were either left untreated (−) or treated with 10 ng ml−1 TNFα for 10 min (+). Cell lysates were prepared, normalized for protein content, and analyzed by immunoblotting with antibodies specific for ASC (top), IκBα (middle), or βActin (bottom). (C) TPA-differentiated THP-1 cells were transfected with ASC (ASC nucleotides 474–494 [+2]) or control (c) siRNA on two consecutive days, then stimulated the following day for 30 min with LPS. Cell lysates were analyzed by immunoblotting for ASC (top), IκBα (middle), or βActin (bottom). (B and C) Blots were also quantified by scanning-densitometry analysis of the autoradiograms, and are presented below the gel as a bar graph showing fold expression compared with uninduced cells, normalized to βActin expression as determined by immunoblotting.
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Related In: Results  -  Collection

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fig5: ASC regulates IκBα levels. (A) Endogenous IκBα expression was analyzed by immunoblotting in stably transfected HEK293N-Neo or ASC-PAAD cells at various times following stimulation with TNFα. The position of unphosphorylated and phosphorylated IκBα proteins are indicated. (B) MCF7 cells were transfected on two consecutive days with two different double-strand ASC siRNAs (ASC nucleotides 721–741 [+1]; and nucleotides 474–494 [+2]) or control (c) siRNA using Oligofectamine. After 24 h, cells were either left untreated (−) or treated with 10 ng ml−1 TNFα for 10 min (+). Cell lysates were prepared, normalized for protein content, and analyzed by immunoblotting with antibodies specific for ASC (top), IκBα (middle), or βActin (bottom). (C) TPA-differentiated THP-1 cells were transfected with ASC (ASC nucleotides 474–494 [+2]) or control (c) siRNA on two consecutive days, then stimulated the following day for 30 min with LPS. Cell lysates were analyzed by immunoblotting for ASC (top), IκBα (middle), or βActin (bottom). (B and C) Blots were also quantified by scanning-densitometry analysis of the autoradiograms, and are presented below the gel as a bar graph showing fold expression compared with uninduced cells, normalized to βActin expression as determined by immunoblotting.
Mentions: As IKK activity induces phosphorylation and subsequent degradation of IκB-family proteins, we evaluated the effects of the PAAD of ASC on levels of endogenous IκBα in these stably transfected cells, before and at various times after TNFα stimulation. Immunoblot analysis of lysates from HEK293N-Neo cells using anti-IκBα antibody demonstrated the appearance of a doublet band (indicative of phosphorylation of IκBα) within 5 min after TNFα simulation, followed by disappearance of IκBα protein. In contrast, IκBα protein levels were sustained at detectable levels in ASC-PAAD–expressing HEK293N cells despite TNFα treatment. Furthermore, the IκBα doublet band indicative of phosphorylation was not observed until much later, at 15–30 min after stimulation (Fig. 5 A), thus demonstrating a marked delay relative to control cells.

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