MKP1 regulates the induction of MUC5AC mucin by Streptococcus pneumoniae pneumolysin by inhibiting the PAK4-JNK signaling pathway

Abstract

Mucosal epithelial cells in the respiratory tract act as the first line of host innate defense against inhaled microbes by producing a range of molecules for clearance. In particular, epithelial mucins facilitate the mucociliary clearance by physically trapping the inhaled microbes. Up-regulation of mucin production thus represents an important host innate defense response against invading microbes. Excess mucin production, however, overwhelms the mucociliary clearance, resulting in defective mucosal defenses. Thus, tight regulation of mucin production is critical for maintaining an appropriate balance between beneficial and detrimental outcomes. Among various mechanisms, negative regulation plays an important role in tightly regulating mucin production. Here we show that the PAK4-JNK signaling pathway acted as a negative regulator for Streptococcus pneumoniae pneumolysin-induced MUC5AC mucin transcription. Moreover pneumolysin also selectively induced expression of MKP1 via a TLR4-dependent MyD88-TRAF6-ERK signaling pathway, which inhibited the PAK4-JNK signaling pathway, thereby leading to up-regulation of MUC5AC mucin production to maintain effective mucosal protection against S. pneumoniae infection. These studies provide novel insights into the molecular mechanisms underlying the tight regulation of mucin overproduction in the pathogenesis of airway infectious diseases and may lead to development of new therapeutic strategies.

FIGURE 1.

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.

FIGURE 2.

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.

FIGURE 3.

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.

FIGURE 4.

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.

FIGURE 5.

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.

FIGURE 6.

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.

FIGURE 7.

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.