Tetradecanoyl phorbol acetate induces expression of Toll-like receptor 2 in U937 cells: involvement of PKC, ERK, and NF-κB

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Abstract

Toll-like receptors (TLRs) have been identified recently as crucial signaling receptors mediating the innate immune recognition. Though induction of TLR2 or TLR4 by 12-O-tetradecanoyl phorbol 13-acetate (TPA) in leukemia cells has been reported, however, the mechanism by which TPA up-regulates TLR2 or TLR4 remains poorly understood. In this study, we investigated the effect of TPA on induction of TLR2 in U937 cells. TPA markedly induced TLR2 mRNA and protein expressions. TLR2 expression in response to TPA was attenuated by pretreatments with GF109203X and Go6976 (inhibitors of protein kinase C (PKC)) and PD98059 (an inhibitor of extracellular signal-regulated kinases (ERKs)), but not SB203580 (an inhibitor of p38s) and SP600125 (an inhibitor of c-Jun N-terminal kinases), suggesting involvement of PKC and ERKs in this response. Moreover, TPA-induced PKC activation was linked to generation of reactive oxygen species, which were dispensable for TLR2 expression in U937 cells. Pretreatments with GF109203X blocked TPA-induced phosphorylation of ERKs, suggesting activation of ERKs by PKC. In addition, TPA induced nuclear factor-κB (NF-κB) activation, which was shown by increased nuclear translocation of p65 NF-κB and degradation of IκB-α, a NF-κB inhibitory protein. Importantly, TPA-induced TLR2 expression was inhibited by blockage of NF-κB activation using NF-κB inhibitors, including MG132 and BAY11-7085. Specifically, TPA-induced nuclear translocation of NF-κB was effectively attenuated by GF109203X and PD98059, suggesting PKC and ERK regulation of NF-κB nuclear localization in response to TPA. Together, these results suggest that TPA-induced TLR2 expression in U937 cells may be at least in part mediated through activation of PKC and ERKs as well as NF-κB transcription factor, and that cross-talk between PKC or ERKs and NF-κB may exist.

Section snippets

Materials and methods

Cell culture. U937 promonocytic leukemia cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2 in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Typically, 3 × 105 cells/ml were seeded in T-25 flasks as 4 ml cultures, and maintained in the tissue culture incubator for 12–16 h before the addition of TPA or other reagents.

Drugs and materials. Antibodies against phospho-ERKs (p-ERKs),

TPA induces TLR2 mRNA expression in U937 cells

First of all, endogenous expression level of TLR2, TLR4, or CD14 was analyzed in human U937, HL60, and K562 cells by RT-PCR using respective primers. THP-1 cells served as positive sources for human TLR2 and TLR4 mRNA expressions. As shown in Fig. 1A, HL60 cells expressed both TLR2 and TLR4 mRNAs. On the other hand, U937 cells expressed only TLR4. K562 cells also expressed very low level of TLR4 mRNA. Expression of CD14 was detected in HL60 and THP-1 cells. TPA has been known to induce

Discussion

Macrophages are patrolling cells of the innate immune system and express TLRs. TLRs are a family of pattern recognition receptors recently identified as crucial signaling receptors mediating the innate immune recognition [3], [4]. Though TLRs may contribute to the resensitization of macrophages to invasive pathogens, however, the mechanism by which TLR expression is regulated in cells is largely unknown. In this study, we have investigated the molecular signaling mechanisms of TLR2 expression

Acknowledgments

This work was supported by Grant No. R13-2002-028-01001-0 from Basic Research Program of Korea Science and Engineering Foundation (KOSEF) to Chronic Diesase Research (CDR) Center at Keimyung University.

References (29)

  • K.A. Zarember et al.

    Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines

    J. Immunol.

    (2002)
  • M. Muzio et al.

    Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells

    J. Immunol.

    (2000)
  • E. Cario et al.

    Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing Toll-like receptors

    J. Immunol.

    (2000)
  • C. Li et al.

    Expression of toll-like receptors 2 and 4 and CD14 during differentiation of HL-60 cells induced by phorbol 12-myristate 13-acetate and 1 alpha, 25-dihydroxy-vitamin D(3)

    Cell Growth Differ.

    (2002)
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