c-Abl-Mediated Tyrosine Phosphorylation of PARP1 Is Crucial for Expression of Proinflammatory Genes

Abstract

Poly(ADP-ribosyl)ation is a rapid and transient posttranslational protein modification mostly catalyzed by poly(ADP-ribose) polymerase-1 (PARP1). Fundamental roles of activated PARP1 in DNA damage repair and cellular response pathways are well established; however, the precise mechanisms by which PARP1 is activated independent of DNA damage, and thereby playing a role in expression of inflammatory genes, remain poorly understood. In this study, we show that, in response to LPS or TNF-α exposure, the nonreceptor tyrosine kinase c-Abl undergoes nuclear translocation and interacts with and phosphorylates PARP1 at the conserved Y829 site. Tyrosine-phosphorylated PARP1 is required for protein poly(ADP-ribosyl)ation of RelA/p65 and NF-κB-dependent expression of proinflammatory genes in murine RAW 264.7 macrophages, human monocytic THP1 cells, or mouse lungs. Furthermore, LPS-induced airway lung inflammation was reduced by inhibition of c-Abl activity. The present study elucidated a novel signaling pathway to activate PARP1 and regulate gene expression, suggesting that blocking the interaction of c-Abl with PARP1 or pharmaceutical inhibition of c-Abl may improve the outcomes of PARP1 activation-mediated inflammatory diseases.

Graphical abstract

FIGURE 1.

Inflammatory agent-stimulated protein PARylation is regulated by c-Abl. (A and B) Inflammatory agents’ (LPS or TNF-α) stimulation promotes protein PARylation. RAW 264.7 cells were challenged with LPS or TNF-α for various lengths of time. Western blotting was performed to detect protein PARylation levels in whole-cell lysates. (C and D) c-Abl activity is required for inflammatory agent-stimulated protein PARylation. RAW 264.7 cells were incubated with or without LPS for 1 h or TNF-α for 3 h in the presence or absence of c-Abl inhibitor (STI571). Cell extracts were subjected to Western blotting to detect the protein PARylation. (E) siRNA of c-Abl blocked protein PARylation. RAW 264.7 cells were transfected with siRNA targeting c-Abl or the control for 48 h and then challenged with LPS for 1 h. Western blotting was performed to detect protein PARylation levels. (F) LPS induces protein PARylation in mice lungs. Mice were challenged with LPS (20 μg per mouse) through the intranasal route for different time intervals, and then the lungs were excised and homogenized. Western blotting was performed to detect the PARylation levels of each protein. (G) LPS-induced PARP1 activation in mice lung is dependent on c-Abl. Mice were challenged with LPS for 1 h with or without i.p. pretreatment of STI571(30 mg·kg⁻¹). Lung homogenates were prepared, and Western blotting was performed to detect protein PARylation levels. Similar results were obtained from at least three independent experiments.

FIGURE 2.

c-Abl interacts with PARP1 in response to exposure to inflammatory agents. (A and B) Exposure to inflammatory agents increases the association of c-Abl with PARP1. RAW 264.7 cells were exposed to LPS (A) or TNF-α (B) for various lengths of time. Whole-cell extracts (WEs) were prepared, and immunoprecipitates were obtained using Ab recognizing PARP1. The association of c-Abl with PARP1 was detected by Western blotting. (C–E) STI571 blocked the association of c-Abl with PARP1. RAW 264.7 cells were mock treated or exposed to LPS (±STI571) for 1 h (C and E) and TNF-α (±STI571) for 3 h (D). WEs were prepared, and immunoprecipitates were obtained using Ab recognizing PARP1 or c-Abl. The blockage in association of c-Abl with PARP1 or vice versa was detected by Western blotting. (F) Diagram of the domains in PARP1 and the schematics of the GST-PARP1 domains expression plasmids. (G) AMD mediates the association of PARP1 with c-Abl. GST and GST-PARP1 domains were incubated with equal amounts of WEs from LPS-treated cells. Levels of pulled-down c-Abl were detected by Western blotting. Similar results were obtained from at least three independent experiments.

FIGURE 3.

LPS stimulation induces c-Abl’s nuclear transportation. (A) LPS stimulation induces nuclear import of c-Abl. RAW 264.7 cells were challenged with LPS for various lengths of time. Cell lysates from nuclear and cytoplasmic fractions were subjected to Western blotting to determine the subcellular distribution of c-Abl. (B) Immune-fluorescence staining verifies LPS-induced nuclear import of c-Abl. RAW 264.7 cells were mock treated or LPS exposed (±STI571) for 1 h, and then cells were fixed and permeabilized and incubated with anti–c-Abl rabbit polyclonal Ab and TRITC-conjugated secondary Ab. The nuclei of the cells were stained with DAPI. Similar results were obtained from at least three independent experiments. Original magnification ×180.

FIGURE 4.

c-Abl is required for tyrosine phosphorylation of PARP1 in response to inflammatory agents. (A and B) Exposure of inflammatory agents increases the levels of tyrosine phosphorylation (pTyr) of PARP1. RAW 264.7 cells were exposed to LPS (A) or TNF-α (B) for various lengths of time. Whole-cell extracts (WEs) were prepared, and immunoprecipitates were obtained using Ab recognizing PARP1. The levels of pTyr of PARP1 were detected by Western blotting. (C and D) Inhibition of c-Abl activity eliminates the inflammatory agent-induced increase in pTyr of PARP1. RAW 264.7 cells were mock treated or exposed to LPS (±STI571) for 1 h (C) or TNF-α exposed (±STI571) for 3 h (D). WEs were prepared, and immunoprecipitates were obtained using Ab recognizing PARP1. The levels of pTyr of PARP1 were detected by Western blotting. (E) siRNA of c-Abl interferes with LPS-induced PARP1 tyrosine phosphorylation. RAW 264.7 cells were transfected with siRNA targeting c-Abl or a control for 48 h and then challenged with LPS for 1 h. WEs were prepared, and immunoprecipitates were obtained using Ab recognizing PARP1. The levels of pTyr of PARP1 in the presence or absence of c-Abl were detected by Western blotting. Similar results were obtained from at least three independent experiments.

FIGURE 5.

PARP1 Y829 might be the site phosphorylated by c-Abl in response to LPS stimulation. (A) Conservative comparison of tyrosine residues of h-PARP1 in segment 751–930 with those relevant in other species through Drosophila to primate. Tyrosine residues that are conserved across all sequences are highlighted with an asterisk (*) and tyrosine residues that are not conserved with a line (—). (B) Y775 and Y829 sites of PARP1 undergo phosphorylation in LPS-exposed cells. HEK 293 cells were transfected with WT Flag-PARP1 as well as Y775F, Y829F, and Y907F mutant plasmids and then challenged with LPS for 1 h. Immunoprecipitates were prepared using flag Ab and then subjected to Western blotting to detect phosphorylation level. (C) The Y829 site of PARP1 might be phosphorylated by c-Abl in response to LPS exposure. WT Flag-PARP1 as well as both Y775F and Y829F mutants were transfected in HEK 293 cells, and then the cells were stimulated with LPS (±STI571) for 1 h. Immunoprecipitates were prepared using Flag Ab and then subjected to Western blotting to detect phosphorylation levels. (D) Y829F mutation does not affect the interaction of c-Abl and PARP1. HEK 293 cells were transfected with WT Flag-PARP1 and Y829F mutant plasmids and then challenged with LPS or left untreated for 1 h. Immunoprecipitates were prepared using Flag Ab and then subjected to Western blotting to detect the interaction with c-Abl. Similar results were obtained from at least three independent experiments.

FIGURE 6.

c-Abl activity promotes RelA/p65 binding with PARP1 and PARyaltion (A and B) c-Abl activity promotes RelA/p65 binding with PARP1. RAW 264.7 cells were mock treated or exposed to LPS (±STI571) for 1 h. Immunoprecipitation was performed, and Ab against PARP1 (A) or RelA/p65 (B) was used to get precipitate complexes. Western blotting was performed using Abs specific for p65 or PARP1 in the complexes, respectively. (C and D) c-Abl activity promotes RelA/p65 PARyaltion. (C) RAW 264.7 cells were treated as described above. Immunoprecipitates were obtained by using Ab against RelA/p65; PARylation levels of RelA/p65 were determined by Western blotting using Ab recognizing PAR. (D) RAW 264.7 cells were transfected with siRNA targeting c-Abl or the control for 48 h and then challenged with LPS for 1 h. Immunoprecipitates were obtained using Ab recognizing RelA/p65. PARylation levels of RelA/p65 in the presence or absence of c-Abl were detected by Western blotting. Similar results were obtained from at least three independent experiments.

FIGURE 7.

Inflammatory agent-induced proinflammatory gene expression is enhanced by c-Abl. (A and B) Inflammatory agents stimulate inflammatory gene expression in murine macrophages. RAW 264.7 cells were incubated with LPS for various lengths of time. Real-time PCR was performed to detect the mRNA expression of TNF-α and IL-1β (n = 5) (A). RAW 264.7 cells were incubated with TNF-α for various lengths of time. Real-time PCR was performed to detect the mRNA expression of Cxcl2 and IL-1β (n = 5) (B). (C and D) c-Abl knockdown blocks upregulation of inflammatory genes. RAW 264.7 cells were subjected to siRNA targeting c-Abl and then mock treated or exposed to LPS for 1 h; real-time PCR was performed to detect the mRNA expression of TNF-α and IL-1β. Inset, Western blotting shows efficacy of c-Abl knockdown (n = 5) (C); c-Abl–deficient RAW 264.7 cells were mock treated or exposed to TNF-α for 3 h, and real-time PCR was performed to detect the mRNA expression of Cxcl2 and IL-1β (n = 5) (D), **p < 0.01. (E) LPS stimulates inflammatory gene expression in mice lungs. Mice were exposed to LPS through the intranasal route for various lengths of time. Mice lungs were collected, and homogenates were prepared. RNA was extracted, and RT-PCR was performed to detect mRNA expression of TNF-α and IL-1β (n = 5). Data were expressed as mean ± SD. Difference significance was analyzed by one-way ANOVA. (F) c-Abl inhibition blocks upregulation of inflammatory genes in mice lungs. Mice were exposed to LPS through the intranasal route for 1 h with or without an i.p. pretreatment of STI571. Mice lungs were collected, and homogenates were prepared. RNA was extracted, and RT-PCR was performed to detect the mRNA expression of TNF-α and IL-1β. Similar results were obtained from at least three independent experiments.

FIGURE 8.

c-Abl activity is responsible for LPS-induced lung inflammation. (A and B) Visual depiction and quantification of cells in BALF. Mice were mock treated or challenged with LPS in the presence or absence of pretreatment with STI571. After 16 h, mice were euthanized, lungs were lavaged, and the cell numbers in BALF were determined. Ten to sixteen randomly selected fields of view per cytospin slide were photographed (A). Differential cell counts were performed after modified Wright–Giemsa staining. Data are average ± SD, representative of six experimental animals from one experimental run. (C) STI571 administration blocked LPS-induced subepithelium accumulation of leukocytes in lung tissues. Mice were treated as described above. Lung tissue sections were processed for staining with H&E to examine the subepithelium accumulation of leukocytes in lung tissues. Similar results were obtained from at least three independent experiments. Original magnification ×200.

FIGURE 9.

Proinflammatory genes’ mRNA expression is impaired in Y829F PARP1-expressing cells. Endogenous PARP1 in Raw 264.7 cells was silenced using siRNA targeting a distinguished sequence of PARP1, and human WT PARP1, Y829F PARP1, and Y907F PARP1 expressional plasmids were transfected and then the cells were stimulated with LPS or not for 1 h. Immunoblotting was performed to detect the interference of endogenous PARP1 as well as the ectopic expression of Flag-tagged WT, Y829F, and Y907F PARP1 (A). Raw 264.7 cells as described above were used; proinflammatory genes’ mRNA levels were detected by RT-PCR and electrophoresis (upper) or real-time PCR (lower) (B). Data were expressed as mean ± SD. Difference significance was analyzed by one-way ANOVA. **p < 0.01.