Protein kinase D1 is essential for MyD88-dependent TLR signaling pathway

Abstract

Protein kinase D1 (PKD1) has been shown to be involved in certain MAPK activation and cytokine expression by several TLR ligands. However, the precise physiological role of PKD1 in individual signaling from TLRs has not been fully addressed. In this study, we provide evidence that PKD1 is being activated by TLR ligands, except the TLR3 ligand. PKD1 activation by TLR ligands is dependent on MyD88, IL-1R-associated kinase 4 and 1, but independent of TNF-alpha receptor-associated factor 6. PKD1-knockdown macrophages and bone marrow-derived dendritic cells revealed that PKD1 is indispensable for the MyD88-dependent ubiquitination of TNF-alpha receptor-associated factor 6; activation of TGF-beta-activated kinase 1, MAPKs, and transcription factors; and expression of proinflammatory genes induced by TLR ligands, but is not involved in expression of type I IFNs induced by TLR ligands and TRIF-dependent genes induced by TLR3 and TLR4 ligands. These results demonstrate that PKD1 is essential for MyD88-dependent proinflammatory immune responses.

Figure 1. TLR ligands induce activation of PKD1 in macrophages

(A) RAW264.7 cells were stimulated with medium (med), CpG DNA (12 μg/ml), LPS (50 ng/ml), Pam3Cys (Pam3CSK(4), 1 μg/ml), PGN (10 μg/ml), FSL-1 (0.1 μg/ml), flagellin (1 μg/ml), imiquimod (10 μg/ml), poly(I:C) (PIC, 50 μg/ml), MDP (10 μg/ml) or PMA (10 ng/ml) for 45 min. (B) RAW264.7 cells were stimulated with poly(I:C) (PIC, 50 mg/ml) or MDP (10 mg/ml) for the indicated time period. CpG DNA (12 mg/ml) or PMA (10 ng/ml) was used as positive control. Activation status of PKDs was detected by Western blot assay using Abs specific for the phosphorylated forms of PKDs (pPKDs744/748, pPKDs916). Phosphorylation of p38 was detected as an indication that each stimulus was functional. (C–D) RAW264.7 cells stably expressing empty vector, FLAG-tagged PKD1, FLAG-tagged PKD2, or FLAG-tagged PKD3 were stimulated as indicated for 45 min. Each PKD family protein in whole cell lysates was immunoprecipitated with anti-FLAG Ab. Kinase activity of PKDs was analyzed by in vitro kinase assay using syntide-2 as a PKD substrate (top). Expression and phosphorylation status of each PKD was analyzed by immunoblotting with anti-FLAG and anti-phospho-PKD Abs, respectively (bottom). (E) Control luciferase-knockdown (Luc shRNA) or PKD1-knockdown (PKD1 shRNA) macrophages were stimulated as indicated for 45 min. Activation status of PKDs was detected by Western blot assay.

Figure 2. TLR ligands induce activation of PKD1 through an MyD88-dependent pathway

Peritoneal macrophages isolated from wild-type, TLR2-gene deficient (TLR2^−/−), TLR4 P712H (Tlr4^Lps-d/Lps-d) mutant, TLR9-gene deficient (TLR9^−/−), MyD88-gene deficient (MyD88^−/−), or TRIF-defective mutant (Trif^Lps2/Lps2) mice were stimulated as indicated for 45 min. Phosphorylation status of PKD1 was detected by Western blotanalysis. Phosphorylation of JNK was detected as an indication that each stimulus was functional.

Figure 3. IRAK4 and IRAK1, but not TRAF6, are required for activation of PKD1 by TLR ligands

(A, B) RAW264.7 cells were stably transfected with vectors expressing control luciferase-shRNA or IRAK4-shRNA under control of the H1 promoter, as described previously (27). Messenger RNA levels (Panel A) and protein levels (Panel B) of the selected molecules in TLR signaling, PKD family, and PKC family in control and IRAK4-knockdown RAW264.7 cells were analyzed by RT-PCR and Western blot assay, respectively. (C, D) Control luciferase-knockdown (Luc shRNA), IRAK4-knockdown (IRAK4 shRNA), or IRAK1-knockdown (IRAK1 shRNA) macrophages were stimulated as indicated. Phosphorylation status of PKD1 was detected by Western blot analysis. (E, F) RAW264.7 cells were stably transfected with vectors expressing control luciferase-shRNA or TRAF6-shRNA under control of the H1 promoter, as described previously (27). Messenger RNA levels (Panel E) and protein levels (Panel F) of the selected molecules in TLR signaling, PKD family, and PKC family in control and TRAF6-knockdown RAW264.7 cells were analyzed by RT-PCR and Western blot assay, respectively. (G) Control luciferase-knockdown (Luc shRNA) or TRAF6-knockdown (TRAF6 shRNA) macrophages were stimulated as indicated. Phosphorylation status of PKD1 was detected by Western blot analysis. Phosphorylation of JNK and TAK1 (pTAK1 Thr184/187) was detected to verify functional effectiveness of TRAF6-knockdown by TRAF6-shRNA.

Figure 4. TLR ligand-mediated MyD88-dependent ubiquitination of TRAF6 is dependent on PKD1

Control (Luc-shRNA) and PKD1-knockdown (PKD1-shRNA) RAW264.7 cells were stimulated as indicated for 1 hr (Panel A) or stimulated with CpG DNA or LPS for indicated time periods (Panels B, C). Whole cell lysates were prepared and immunoprecipitated with anti-TRAF6 Ab and the resulting immunoprecipitates were analyzed by Western blot using anti-ubiquitine (UBQ) to detect the ubiquitinated form of TRAF6.

Figure 5. PKD1 is required for MyD88-dependent activation of TAK1, MAPKs and transcription factors

(A–F) Control luciferase-knockdown (Luc shRNA) or PKD1-knockdown (PKD1 shRNA) macrophages were stimulated as indicated for 45 min (Panels A, C, E), 1 hr (Panel E, for NF-κB) or 4 hr (Panel E, for AP-1), or stimulated with LPS for indicated time periods (Panels B, D, F). Phosphorylation of TAK1, JNK, p38, ERK, CREB, and STAT1 was detected by Western blot analysis. DNA-binding activities of transcription factor, NF-κB or AP-1, in equal amounts of nuclear extracts (3 μg/lane) were analyzed by EMSA (gel-shift) and degradation of IκBα and IκBβ in cytosolic extracts was detected by Western blot analysis (WB). (G) Cells were transiently transfected with AP-1-β-galactosidase, or pRL-TK plus NF-κB-luciferase, CREB- luciferase or ISRE-luciferase reporter genes and then stimulated as indicated for 12 h. Luciferase (NF-κB, CREB or ISRE) or β-galactosidase (AP-1) activities in cell extracts were analyzed. Data represent the mean relative luciferase unit (RLU) ±SD of triplicates.

Figure 6. PKD1 is essential for the expression of MyD88-dependent genes, but dispensable for the expression of TRIF-dependent genes in RAW264.7 cells

Control luciferase-knockdown or PKD1-knockdown RAW264.7 cells were stimulated as indicated for 24 h (Panels A, D) or 4 h (Panels B, C). Levels of the indicated proteins in the culture supernatant (Panel A) and mRNA (Panels B, C) were analyzed by ELISA and RT-PCR, respectively. Surface expression levels of CD86 were detected by flow cytometric analysis (Panel D). ELISA data represent the mean cytokine concentration (pg/ml) ±SD of triplicates.

Figure 7. PKD1 is essential for the expression of MyD88-dependent genes, but dispensable for the expression of TRIF-dependent genes in bone-marrow derived dendritic cells

(A, B) BMDCs were transiently transfected with 100 nM of non-target siRNA (NT siRNA) or PKD1-sepcific siRNA (PKD1 siRNA) using lipofectamine. Messenger RNA levels (Panel A) and protein levels (Panel B) of the selected molecules in TLR signaling, PKD family, and PKC family in control and PKD1-knockdown BMDCs were analyzed by RT-PCR and Western blot assay, respectively. (C–E) Control or PKD1-knockdown BMDCs were stimulated as indicated for 24 h (Panels C, E) or 4 h (Panel D). Levels of the indicated proteins in the culture supernatant (Panel C) and mRNA (Panel D) were analyzed by ELISA and RT-PCR, respectively. Surface expression levels of CD86 were detected by flow cytometric analysis (Panel E). ELISA data represent the mean cytokine concentration (pg/ml) ±SD of triplicates.

Figure 8. Effect of pharmacological PKC/PKD inhibitor Gö6976 on TLR ligand-mediated cytokine and chemokine expression

C57BL/6 mice were injected intraperitoneally with DMSO or Gö6976 (2.5 mg/kg body weight) at 4 hr and 1 hr before the TLR ligand stimulation. DMSO or Gö6976 pretreated mice were injected intraperitoneally with PBS, CpG DNA (30 μg/mouse), LPS (2 μg/mouse) or PIC (150 μg/mouse). Two hr later, mice were bled to obtain serum and then euthanized. Spleen was isolated. Total spleen RNA and whole spleen cell lysates were prepared. Phosphorylation of PKD1, JNK, p38 and ERK and presence of IκBα and IκBβ in whole spleen cell lysates were detected by Western blot analysis (Panel A). Messenger RNA levels of the indicate cytokines and chemokines in spleen were analyzed by RT-PCR (Panel B). Levels of the indicated cytokines in serum were analyzed by cytokine-specific ELISAs. Data represent the mean concentration (pg/ml) ±SD of triplicates.

Figure 9. Hypothetical model for PKD1 involvement in TLR/MyD88 signaling pathway

Ligand-bound TLR recruits MyD88. TLR-bound MyD88 interacts with several proteins including IRFs, TRAF3, IRAK4 and IRAK1. On one hand, activated IRFs and TRAF3 initiates signaling cascades that lead to production of type I IFNs. One the other hand, IRAK1 is activated by IRAK4 in the TLR/MyD88/IRAK receptor complex. Subsequently, PKD1 is recruited to the TLR/MyD88 receptor complex by interacting with IRAK4 and IRAK1 and then activated by IRAKs. IRAK1-bound activated PKD1 leaves the TLR/MyD88 receptor complex and binds to TRAF6, which allows TRAF6 to interact with IRAK1, and leads to the ubiquitination of TRAF6. Ubiquitinated TRAF6 leads to the activation of TAK1, which in turn activates signaling cascades that lead to activation of MAPKs and NF-κB, and subsequent production of pro- inflammatory cytokines and chemokines.