The NLR adaptor ASC/PYCARD regulates DUSP10, mitogen-activated protein kinase (MAPK), and chemokine induction independent of the inflammasome - PubMed (original) (raw)

The NLR adaptor ASC/PYCARD regulates DUSP10, mitogen-activated protein kinase (MAPK), and chemokine induction independent of the inflammasome

Debra J Taxman et al. J Biol Chem. 2011.

Abstract

ASC/PYCARD is a common adaptor for a diverse set of inflammasomes that activate caspase-1, most prominently the NLR-based inflammasome. Mounting evidence indicates that ASC and these NLRs also elicit non-overlapping functions, but the molecular basis for this difference is unclear. To address this, we performed microarray and network analysis of ASC shRNA knockdown cells. In pathogen-infected cells, an ASC-dependent interactome is centered on the mitogen-activated protein kinase (MAPK) ERK and on multiple chemokines. ASC did not affect the expression of MAPK but affected its phosphorylation by pathogens and Toll-like receptor agonists via suppression of the dual-specificity phosphatase, DUSP10/MKP5. Chemokine induction, DUSP function, and MAPK phosphorylation were independent of caspase-1 and IL-1β. MAPK activation by pathogen was abrogated in Asc(-/-) but not Nlrp3(-/-), Nlrc4(-/-), or Casp1(-/-) macrophages. These results demonstrate a function for ASC that is distinct from the inflammasome in modulating MAPK activity and chemokine expression and further identify DUSP10 as a novel ASC target.

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Figures

FIGURE 1.

FIGURE 1.

Network analysis of _Pg_-stimulated genes in control and ASC-knockdown cells. A–C, interactomes identified by Ingenuity analysis of the GeneSpring 7.0 microarray genes in

supplemental Table S3

. Values represent -fold stimulation for _Pg_-stimulated versus control THP1 cells. Positive values and red color represent increase in _Pg_-stimulated cells, while green color and negative values represent decrease. Bold lines represent physical interactions between proteins, and dotted lines represent functional interactions. A, cell to cell signaling interactome, p < 10E-46; B, cellular growth and proliferation interactome, p < 10E-46; C, gene expression interactome, p < 10E-41. D, assessment of ASC knockdown in shASC THP1 cell lines. Control cells include THP1 and THP1 expressing an empty vector (EV) or a mutated target site (shASC#1mut). ASC-knockdown cell lines, shASC#1 and -#2, encode different shRNAs for ASC. ASC expression was measured by real-time PCR and normalized to an average of 100 in control cell lines. Data represent averages ± S.D. for three independent experiments. E, IL-1β ELISA of supernatants from control and ASC-knockdown cells following Pg infection. Data represent averages ± S.D. for three independent experiments. F, Ingenuity analysis of genes differentially activated by Pg in the presence and absence of ASC. The “inflammatory and immunological disease” interactome depicted (p < 10E-42) was based on GeneSpring 7.0 microarray values from

supplemental Table S4

. Values represent -fold stimulation for _Pg_-infected shASC#1 versus shASC#1mut cells. Negative values and green color represent a decrease in shASC#1 cells, while positive values and red color represent an increase.

FIGURE 2.

FIGURE 2.

Chemokine transcription is reduced in shASC-containing THP1 cell lines. A, TNFA expression over a time course of infection with Pg. Real-time PCR values were normalized to 1 in uninfected THP1 cells. Representative of three independent experiments. B–D, expression of CCL3, CCL4, and CXCL3 as measured by real-time PCR. Data were normalized to 100 in _Pg_-induced THP1 cells. N.D., not detectable. Data represent averages ± S.D. for at least three independent experiments. E, RNA decay following DRB treatment as measured by real-time PCR. Starting values were normalized to 100. Data represent averages ± S.D. and are representative of three independent experiments. F, real-time PCR of nascent transcripts in control and shASC cells. Data were normalized to 1 in uninfected cells. Representative of three independent experiments.

FIGURE 3.

FIGURE 3.

Analysis of secreted cytokine and chemokine levels in control and shASC-containing THP1 lines. A, -fold induction of cytokines and chemokines in supernatants 24 h following Pg infection. Values represent ratios of binding on RayBio® Human Cytokine Antibody Array 5 chips for 3-fold or more difference between shASC #1mut and shASC#1 cells. B–E, ELISA of TNF-α, CCL3, CCL20, and IGF-1 prior to or following 24-h infection with Pg. Data represent averages ± S.D. for at least three independent experiments.

FIGURE 4.

FIGURE 4.

MAPK activation is reduced in shASC-containing THP1 cells. A, Western analysis of p-ERK, total ERK (ERK1 and ERK2), and GAPDH in shASC#1mut and shASC#1 cells following a time course of infection with Pg. Representative of at least five independent experiments. B, Western analysis of p-ERK and GAPDH in shASC#1mut and shASC#1 cells following 60-min treatment with E. coli or TLR agonist. Representative of three independent experiments. P3C, Pam3Cys-Ser-(Lys)4-trihydrochloride. C, Western analysis of p-JNK and total JNK in shASC#1mut and shASC#1 cells following a time course of infection with Pg. Representative of at least three independent experiments. D, ELISA of chemokine levels in THP1 cells treated with Pg, DMSO solvent, ERK, and/or JNK inhibitor. Data represent average ± S.D. for at least three independent experiments. N.D., not detectable. E, ELISA of IGF-1 levels in _Pg_-treated cells following addition of DMSO, ERK, or JNK inhibitor. Data represent averages ± S.D. for three independent experiments.

FIGURE 5.

FIGURE 5.

MAPK activation and chemokine induction in primary mouse macrophages is Asc-dependent. A–C, Western blot of p-ERK, p-JNK, and p-p38 in primary mouse macrophages from WT C57BL/6 and _Asc_−/− mice following a time course of infection with Pg. Blotting for the Asc protein is shown as a verification of the knockout, and GAPDH is shown as a loading control. Representative of at least three independent experiments. D, chemokines modulated in _Asc_−/− mice as assessed by pathway-focused gene expression profiling of pooled RNA from six mice. Values represent -fold expression for _Pg_-infected _Asc_−/− versus WT macrophages. Negative values represent a decrease in _Asc_−/− macrophages, and positive values represent an increase. A complete list of genes is provided in

supplemental Table S5

. E, induction of chemokines following 2-h Pg infection as assessed by TaqMan® PCR. Data represent averages ± S.D. for four independent experiments; *, p < 0.05; **, p < 0.05. F, effects of MAPK inhibitors on Ccl3 expression as determined by TaqMan® Assays. Expression levels were normalized to 100 in control cells. Data represent averages ± S.D. for three independent experiments.

FIGURE 6.

FIGURE 6.

Elevated DUSP10 levels negatively regulate _Pg_-induced chemokine expression. A, real-time analysis of DUSP10 and MAPK1/ERK2 RNA in control and shASC-containing cell lines following 2-h Pg infection. Expression was normalized to 1 in THP1 cells. Representative of at least three independent experiments. B and C, real-time PCR of DUSP10 RNA over a time course of infection with Pg for (B) shASC#1mut versus shASC#1 THP1 cells and (C) WT C57BL/6 _versus Asc_−/− murine primary macrophages. Expression was normalized to 1 in uninfected control cells. Data represent averages ± S.D. for three independent experiments. D, real-time PCR of DUSP10 RNA levels in THP1, pLex-EV, and pLex-DUSP10 cells. Values were normalized to 1 in THP1 cells. Data represent average ± S.D. for three independent experiments. E, Western analysis of p-ERK and p-JNK in non-transfected THP1 cells and pLex-EV- and pLex-DUSP10-containing cells following a time course of infection with Pg. This was representative of three independent experiments. F, ELISA of CCL3 and CCL20 in supernatants from cells expressing pLex-EV or pLex-DUSP10 18 h following infection with Pg. N.D., not detectable. Data represent averages ± S.D. for three independent experiments. G, real-time PCR of ASC and DUSP10 RNA levels in single or double knockdown THP1 cells created by transduction with lentivirus expressing shASC#1 or shASC#1mut and shDUSP10 (pLKO-shDUSP10) or an empty vector (pLKO). Values were normalized to an average of 100 in control cells. Data represent average ± S.D. for three independent experiments. H, Western analysis of p-ERK and p-JNK in shASC#1mut- and shASC#1-containing THP1 cells transduced with pLKO control or pLKO-shDUSP10-expressing lentivirus. This was representative of three independent experiments. I and J, real-time PCR analysis of DUSP10 expression following pretreatment with MAPK inhibitors in THP1 cells (I) and primary mouse macrophages (J). TNFA is shown as a control. Expression was normalized to 1 in control cells. Data represent averages ± S.D. for three independent experiments; *, p < 0.05.

FIGURE 7.

FIGURE 7.

Induction of chemokines and ERK phosphorylation by Pg is IL-1β-, caspase-1-, and NLRP3-independent. A, real-time PCR of chemokine RNA in THP1 cells treated with Pg and/or the IL-1 receptor antagonist, Kineret®. Data are normalized to an average of 100 in _Pg_-treated THP1 cells and represent averages ± S.D. for three independent experiments. N.D., not detectable. B, ELISA of IL-1β, CCL3, and CCL20 in supernatant from THP1 cells treated with Pg, Kineret®, and/or the caspase-1 inhibitor YVAD-cmk. C and D, Western analysis of p-ERK in THP1 cells following a time course of infection with Pg. Kineret® (C) or YVAD-cmk (D) was added as indicated. GAPDH is shown as a loading control. Representative of three independent experiments. E, caspase-1 activation in shASC#1 and pLex-DUSP10 cells following Pg infection. Immunoprecipitation/immunoblotting was performed in uninfected cells (lanes 1–4) and following 2.5-h Pg infection (lanes 5–8). An immunoblot for actin in cell lysates is shown as a loading control. Representative of three independent experiments. F–I, Western blot of p-ERK following a time course of infection with Pg in primary mouse macrophages from WT C57BL/6 _versus MyD88_−/−, _Casp1_−/−, _Nlrp3_−/−, or _Nlrc4_−/− mice. GAPDH is shown as a loading control. Representative of three independent experiments. J, Western blot of p-ERK following a time course of infection with Pg in primary mouse macrophages. Supernatant from _Pg_-infected WT or _ASC_−/− mouse macrophages was applied to WT and _ASC_−/− mouse macrophages 5 min prior to infection.

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