PARP-1 deficiency blocks IL-5 expression through calpain-dependent degradation of STAT-6 in a murine asthma model - PubMed (original) (raw)

PARP-1 deficiency blocks IL-5 expression through calpain-dependent degradation of STAT-6 in a murine asthma model

R Datta et al. Allergy. 2011 Jul.

Abstract

Background: We recently showed that poly(ADP-ribose)polymerase-1 (PARP-1) may play a role in allergen (ovalbumin)-induced airway eosinophilia, potentially through a specific effect on IL-5 production. We also reported that while IL-5 replenishment promotes reversal of eosinophilia in lungs of PARP-1(-/-) mice, IL-4 or Immunoglobulin E replenishment do not, suggesting a potentially significant regulatory relationship between PARP-1 and IL-5.

Objective: To explore the mechanism by which PARP-1 regulates IL-5 production and to determine how PARP-1 inhibition blocks allergen-induced eosinophilia.

Methods: This study was conducted using a murine model of allergic airway inflammation and primary splenocytes.

Results: PARP-1 knockout-associated reduction in IL-5 upon allergen exposure occurs at the mRNA level. Such an effect appears to take place after IL-4 receptor activation as PARP-1 inhibition exerted no effect on JAK1/JAK3 activation. Signal transducer and activator of transcription-6 (STAT-6) protein was severely downregulated in spleens of PARP-1(-/-) mice without any effect on mRNA levels, suggesting an effect on protein integrity rather than gene transcription. Interestingly, the degradation of STAT-6 in PARP-1(-/-) mice required allergen stimulation. Additionally, PARP-1 enzymatic activity appears to be required for STAT-6 integrity. The downregulation of STAT-6 coincided with mRNA and protein reduction of GATA-binding protein-3 and occupancy of its binding site on the IL-5 gene promoter. IL-4 was sufficient to induce STAT-6 downregulation in both PARP-1(-/-) mice and isolated splenocytes. Such degradation may be mediated by calpain, but not by proteasomes.

Conclusion: These results demonstrate a novel function of PARP-1 in regulating IL-5 expression during allergen-induced inflammation and explain the underlying mechanism by which PARP-1 inhibition results in IL-5 reduction.

© 2011 John Wiley & Sons A/S.

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Figures

Figure 1

Figure 1. PARP-1 gene deletion-associated inhibition of IL-5 occurs at the mRNA level without an effect on IL-4R activation

(A) WT and PARP-1−/− mice were sensitized to and challenged with OVA. Mice were then sacrificed and lungs were subjected to BAL and spleens were collected for RNA or protein extraction. (A) BAL fluids were assessed for IL-5 using a BioRad single-plex system. Data are given as means ± SEM of values obtained from at least six mice per group. *, difference from unchallenged mice, p < 0.01; #, difference from WT mice subjected to the OVA challenge, p < 0.01. (B) Total RNA, extracted from portions of the collected spleens, was subjected to cDNA generation followed by conventional (upper panels) or real-time (bottom panel) PCR with primers specific to murine IL-5 or β-actin. (C) Protein extracts were prepared from the remaining portions of the collected spleens and subjected to immunoblot analysis with antibodies to JAK1, JAK3, the phosphorylated form of JAK1 at tyrosine residue 1034 (p1034-JAK1), the phosphorylated form of JAK3 at tyrosine residue 785 (p785-JAK3), or actin. Note that JAK1 and JAK3 blots (C, bottom panels) are of the same samples used for p1034-JAK1 and p785-JAK3, respectively but were generated using a different gel. The immunoblots were quantified using Adobe Photoshop CS and data is expressed as relative density; *, Difference from untreated WT control, p< 0.01.

Figure 2

Figure 2. PARP-1 gene deletion promotes STAT-6 protein degradation in spleens in an allergen-dependent manner with a consequent reduction in GATA-3 expression

Spleens from the different experimental groups were subjected to total or nuclear protein or RNA extraction. Total protein extracts were then subjected to immunoblot analysis with antibodies to STAT-6 or actin (A) or to its phosphorylated form on tyrosine residue 641 (p641-STAT-6) (B); the immunoblots were quantified using Adobe Photoshop CS and data is expressed as relative density; *, difference from untreated control, p< 0.05; #, difference from OVA-treated WT mice, p< 0.05. (C) Nuclear extracts were subjected to EMSA using a radiolabeled oligonucleotide containing the STAT-6 consensus sequence. (D) Mice received an i.p. injection of TIQ-A (6 mg/kg) or vehicle alone 1 h prior OVA challenge. Mice were sacrificed 24 h later. Spleens were removed for protein extraction after which proteins were subjected to immunoblot analysis with antibodies to STAT-6 or actin; extracts from spleens of OVA-challenged PARP-1−/− mice were used as a control for STAT-6 degradation. The immunoblots were quantified as above and data is expressed as relative density; *, difference from OVA-treated WT mice, p< 0.05. Total RNA was subjected to cDNA generation followed by conventional (upper panels) or real-time (lower panels) PCR with primers specific to STAT-6 (E) or GATA-3 (F); β-actin was used as an internal control. *, difference from OVA-treated WT mice, p< 0.01. (G) Protein extracts were subjected to immunoblot analysis with antibodies to murine GATA-3 or actin.

Figure 3

Figure 3. IL-4 promotes a severe reduction in STAT-6 protein levels spleens of treated PARP-1−/− mice and in vitro in treated PARP-1−/− splenocytes

WT (A) and PARP-1−/− (B) mice received a single i.p. injection of IL-4 (100ng/mouse). Mice were sacrificed 6 or 12 h later and spleens were removed and processed for protein isolation. Proteins were then subjected to immunoblot analysis with antibodies to STAT-6 or actin; the immunoblots were quantified using Adobe Photoshop CS and data is expressed as relative density; *, difference from untreated control, p< 0.05. Splenocytes were isolated from naive WT or PARP-1−/− mice and stimulated with IL-4 (10ng/ml) for 12 hours. Cells were lysed then subjected to total protein or RNA extraction. A portion of the cells was also subjected to cross-linking with formaldehyde as described in the Methods. (C) Protein extracts were subjected to immunoblot analysis with antibodies to STAT-6 or actin. The immunoblots were quantified and data is expressed as relative density; *, difference from untreated control, p< 0.05; #, difference from IL-4-treated WT mice, p< 0.05. (D) Total RNA was subjected to cDNA generation followed by conventional PCR with primers specific to mouse IL-5; β-actin was used as an internal control. (E) Formaldehyde-fixed cells were subjected to ChIP assay using antibodies to GATA-3. Levels of immunoprecipitated chromatin fragments (−70 to −59 region) of the mouse IL-5 gene promoter input were examined by PCR.

Figure 4

Figure 4

Calpain inhibition blocks degradation of STAT-6 in PARP-1−/− splenocytes upon IL-4 stimulation. Splenocytes isolated from WT or PARP-1−/− mice were treated with IL-4 in the absence or presence of 20 µM of the proteasome inhibitor MG132 or the calpain inhibitor ALLN. Cells were collected 12 h after treatment and subjected to protein extraction. Proteins were then subjected to immunoblot analysis with antibodies to STAT-6 or actin. (B) The immunoblot results in A were quantified and expressed as STAT-6 relative (over actin). *Difference from the control that received neither IL-4 nor protease inhibitors; #, Difference from IL-4-treated cells without protease inhibitors, p< 0.01. (C) Lysates from control cells that were treated with MG132 or left untreated were subjected to immunoblot analysis with antibodies to ubiquitin.

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