PAK4 acts upstream of JNK in negative regulation of MUC5AC induction by pneumolysin. A, purified recombinant pneumolysin (Ply) potently induced phosphorylation of PAK4 in a time-dependent manner as assessed by performing Western blot analysis in HMEEC-1 cells. B, overexpressing a dominant-negative PAK4 enhanced recombinant pneumolysin-induced MUC5AC expression at transcriptional levels in HMEEC-1 and HeLa cells, whereas overexpressing a constitutively active form of PAK4 (PAK4 CA) inhibited it. C, PAK4 knockdown by using siRNA-PAK4 (100 nm) enhanced MUC5AC expression at mRNA levels by recombinant pneumolysin (right panel) in HeLa cells. The efficiency of siRNA-PAK4 in reducing endogenous PAK4 mRNA was confirmed by performing real time Q-PCR (left panel). D, phosphorylation of JNK by recombinant pneumolysin was reduced by overexpressing a dominant-negative PAK4 in HeLa cells. Data in A and D are representative of three separate experiments. Data in B and C are expressed as mean ± S.D. (n = 3). ^*, p < 0.05 versus mock in the presence of recombinant pneumolysin (B and C). ^**, p < 0.01 versus control group (C). p-PAK4, phosphorylated PAK4; p-JNK, phosphorylated JNK; CON, control.

MKP1 acts as a positive regulator for pneumolysin-induced MUC5AC mucin transcription. A, 2.5 μm Ro31-8220 reduced recombinant pneumolysin (Ply) (100 ng/ml)-induced MUC5AC transcription in HeLa cells. B, overexpressing a dominant-negative MKP1 inhibited recombinant pneumolysin-induced MUC5AC expression at transcriptional levels in HeLa cells, whereas overexpressing a wild-type MKP1 enhanced it. C, MKP1 knockdown by using siRNA-MKP1 (100 nm) reduced MUC5AC induction at mRNA levels by recombinant pneumolysin (right panel) in HeLa cells. The efficiency of siRNA-MKP1 in reducing endogenous MKP1 mRNA was confirmed by performing real time Q-PCR (left panel). D, overexpressing wild-type MKP1 reduced recombinant pneumolysin-induced JNK phosphorylation. E, MKP1 knockdown by using siRNA-MKP1 (100 nm) increased JNK phosphorylation by recombinant pneumolysin (lower panel) in HeLa cells. The efficiency of siRNA-MKP1 in reducing endogenous MKP1 protein was confirmed by performing Western blot analysis (upper panel). Data in A–C are expressed as mean ± S.D. (n = 3). Data in D and E are representative of three separate experiments. ^*, p < 0.05 versus in the presence of only recombinant pneumolysin (A). ^*, p < 0.05 versus mock in the presence of recombinant pneumolysin (B and C). ^**, p < 0.01 versus control group (C). CON, control; p-JNK, phosphorylated JNK.

TLR4-MyD88-TRAF6-ERK signaling is required for MKP1 induction by pneumolysin. A, overexpressing a dominant-negative TLR4 reduced recombinant pneumolysin (Ply)-induced MKP1 expression at mRNA levels in HMEEC-1 and HeLa cells. B, overexpressing wild-type TLR4 in Tlr4^Lps-d MEFs markedly enhanced Mkp1 expression at the mRNA level. C, induction of MKP1 by recombinant pneumolysin (100 ng/ml) was reduced in Tlr4^Lps-d MEF cells compared with wild-type MEFs. D, overexpressing a dominant-negative MyD88 and TRAF6 reduced recombinant pneumolysin-induced MKP1 expression at mRNA levels in HeLa cells. E and F, overexpressing a dominant-negative MyD88 (E) and TRAF6 (F) reduced expression of MKP1 in HeLa cells. G, overexpressing a dominant-negative ERK1 reduced recombinant pneumolysin-induced MKP1 expression at mRNA levels in HeLa cells. Data in A, B, D, and G are expressed as mean ± S.D. (n = 3). Data in C, E, and F are representative of three separate experiments. ^*, p < 0.05 versus mock in the presence of recombinant pneumolysin (A, B, D, and G). CON, control.

JNK acts as a negative regulator for pneumolysin-induced MUC5AC mucin transcription. A, purified recombinant pneumolysin (Ply) induced phosphorylation of JNK in a time-dependent manner as assessed by performing Western blot analysis in HMEEC-1 cells. B, 1 μm SP600125 enhanced recombinant pneumolysin (100 ng/ml)-induced MUC5AC transcription in HeLa cells. C, overexpressing DN JNK1 and JNK2 enhanced pneumolysin-induced MUC5AC expression at transcriptional levels in HMEEC-1, HeLa, and HM3 cells. D, JNK1 or JNK2 knockdown by using siRNA-JNK1 or -2 (100 nm) enhanced MUC5AC expression at mRNA levels by pneumolysin (right panels) in HeLa cells. The efficiency of siRNA-JNK1 or -2 in reducing endogenous JNK1 or JNK2 mRNA was confirmed by performing real time Q-PCR (left panel). E, JNK1 or JNK2 knockdown by using siRNA-JNK1 or -2 (100 nm) enhanced MUC5AC expression at protein levels by pneumolysin (right panel) in HMEEC-1 cells. MUC5AC protein was measured by enzyme-linked immunosorbent assay and expressed as percentage above base line. The efficiency of siRNA-JNK1 or -2 in reducing endogenous JNK1 or JNK2 mRNA was confirmed by performing real time Q-PCR (left panel). F, SP600125 (0.25 mg/kg intraperitoneally) enhanced S. pneumoniae lysate-induced Muc5ac mRNA expression in the middle ear of C57BL/6 mice in vivo. Data in A are representative of three separate experiments. Data in B–F are expressed as mean ± S.D. (n = 3). ^*, p < 0.05 versus in the presence of only recombinant pneumolysin (B). ^*, p < 0.05 versus mock in the presence of recombinant pneumolysin (C–E). ^*, p < 0.05 versus in the presence of only S. pneumonia (F). ^**, p < 0.01 versus control group (D and E). p-JNK, phosphorylated JNK; CON, control.

Pneumolysin induces activation of PAK4-JNK pathway via TLR4-MyD88-TRAF6. A–C, overexpressing a dominant-negative TLR4 (A), MyD88 (B), and TRAF6 (C) reduced phosphorylation of PAK4 and JNK in HeLa cells. Data in A–C are representative of three separate experiments. p-PAK4, phosphorylated PAK4; p-JNK, phosphorylated JNK.

MKP1 expression is induced by pneumolysin. A, recombinant pneumolysin (Ply) (100 ng/ml) induced MKP1 but not MKP3, -5, and -7 at the mRNA level in HeLa cells. B, MKP1 transcription was induced by D39 WT and recombinant pneumolysin (100 ng/ml) but not by pneumolysin-deficient mutant (Ply mt) in HMEEC-1 cells. C, D39 WT induced Muc5ac mRNA expression in the middle ear of C57BL/6 mice in vivo. D, purified recombinant pneumolysin induced MKP1 expression as assessed by performing Western blot analysis in HMEEC-1 cells. Data in A–C are expressed as mean ± S.D. (n = 3). Data in D are representative of three separate experiments. ^*, p < 0.05 versus control group (A–C). ^**, p > 0.05 versus control group (A and B). CON, control.

Schematic representation of tight regulation of MUC5AC mucin induction by pneumolysin via PAK4-JNK and MKP1 signaling pathways. As indicated, the PAK4-JNK signaling pathway negatively regulates MUC5AC induction by recombinant pneumolysin in a TLR4-dependent manner. Interestingly induction of MKP1 appears to be crucial for inhibiting JNK activation. Our data thereby unveil a complex signaling mechanism underlying tight regulation of MUC5AC mucin by pneumococcal pneumolysin.