Phosphorylation Of Heat Shock Protein 27 (HSP27) Regulates Downstream Inflammatory Signaling Pathways By Influencing The Transport Of Toll-like Receptor 4 (TLR4)

Heat shock protein 27 (HSP27) is a member of the small Hsps family expressed in different adult cell types. In addition to its protective function for proper protein folding in heat shock and other conditions of stress (free radicals, Ischemia, toxic substance), HSP27 has been linked to different signaling pathways regulating critical cellular functions such as development, proliferation, apoptosis, differentiation, and actin remodeling. Most of the previous studies focused on the regulatory role of HSP27 protein itself in various signaling pathways; however, the effect of its phosphorylation state is unclear. This research focuses on the regulatory roles of HSP27 and its phosphorylated form on Toll-like receptor 4 (TLR4) and inflammatory signaling pathways.Along with previous work of our group, we confirmed HSP27 was phosphorylated in the LPS-induced inflammatory response. In preliminary experiment considerable HSP27 expression was observed in human acute monocytic leukemia cell line THP1. After LPS stimulus, Ser15 and Ser78 residues underwent a significant phosphorylation and dephosphorylation process within 2 h, whereas Ser82 residue remained low-level expression. We applied three aspects of methods to control intracellular HSP27 phosphorylation in various experiments. First, two synthetic agents were used, specific HSP27 phosphorylation inhibitor and p38 MAPK (upstream kinase of HSP27) inhibitor SB203580. Second, we did RNA interference for MK2, which is main direct upstream kinase for HSP27. Third, HSP27 expression vector with a three-residue mutation (S15/78/82) of the protein was used to mimic phosphorylated form in cells. In THP1 cells, inhibitors (KRIBB3 and SB203580) and MK2 RNAi could significantly downregulated TLR4 ligand LPS induced the release of cytokines, as well as the expression of inflammation hallmark enzymes iNOS and COX-2. Furthermore, both of TLR4 downstream pathways, MyD88-dependent NF-kB pathway and MyD88-independent/TRIF-dependent IRF3 pathway were attenuated by HSP27 phosphorylation inhibitors and MK2 RNAi. However, when THP1 cells were challenged by the ligand of TLR3—oly (I:C), activation of IRF3 pathway was not impaired under inhibiting HSP27 phosphorylation. Phagocytosis is also a hallmark of inflammation intensity, which is enhanced under LPS stimulation in monocytes/macrophages. Morphological and flow cytometric data indicated that KRIBB3 and SB203580 could weaken phagocytosis. We thus established endotoxin shock animal model to investigate therapeutic effect of KRIBB3 for acute inflammation and survival ratio of high-dose LPS-challenged mice was significantly increased by KRIBB3.Both in cells and endotoxic model, anti-inflammatory effect of HSP27 phosphorylation inhibition were considerable, indicating a consequential mechanism Since HSP27 phosphorylation regulated MyD88-dependent and MyD88-independent pathways at the same time but not TLR3 ligand activated IRF3 pathway, the target for phosphorylated HSP27 was definitely upstream of the signaling pathway. Additionally, it is reported that phagocytosis ability of monocytes/macrophages was tightly related to TLRs expression, thus we investigated the LPS receptors TLR4 and TLR2. Flow cytometric data demonstrated that TLR2 cell surface localization maintained a stable amount under LPS-challenge and was not related to HSP27 phosphorylation inhibitors; but LPS-stimulated TLR4 was up regulated and succedent downregulated, which was eliminated by KRIBB3 and SB203580. According to previous research, besides cell membrane, Golgi apparatus was an important cellular compartment for TLR4 prior to trafficking to membrane. We used Golgi-specific antibody to stain the organelle, fluorescence data showed antibody-labeled TLR4 trafficking was blocked by HSP27 phosphorylation inhibitors. Interestingly, other immunofluorescence and confocal observation showed HSP27 partly translocated to Golgi apparatus, which is similar with reported phenomenon in other inflammation cell line (J774.1). Above data indicated inhibition of HSP27 phosphorylation influenced TLR4 trafficking and cell surface localization. We further demonstrated HSP27 could interact with TLR4 through immunoprecipitation, and that interaction was enhanced after LPS stimulus. Experiments with transfection of phosphomimetic mutant, as well as consecutive immunoprecipitation, confirmed that only phosphorylated HSP27 interacts with TLR4. On the other hand, the process that TLR4 trafficking from Golgi apparatus to cell membrane needs TLR4 glycosylation for mature TLR4 formation. Interestingly, we found KRIBB3 could partly inhibit TLR4 glycosylation.In conclusion, our study reveals a novel role of phosphorylated HSP27 as an regulatory factor in cellular inflammatory response to LPS and elucidates a new mechanism that HSP27 phosphorylation regulates TLR4 maturation and trafficking via directly interacting with this molecule affecting its glycosylation, which provides a new insight for penetratingly analyzing the role of this potentiated drug target in inflammation and pharmacological research.