Recombinant human KAI1/CD82 attenuates M1 macrophage polarization on LPS-stimulated RAW264.7 cells via blocking TLR4/JNK/NF-κB signal pathway
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KAI1/CD82, a membrane tetraspanin protein, can prevent variouscancers and retinal disorders through its anti-angiogenicand anti-metastatic capacity. However, little is known about itsanti-inflammatory effect and molecular mechanism. Therefore,the present study aimed to inLPSvestigate effect of a recombinantprotein of the large extracellular domain of human KAI1(Gly 111-Leu 228, rhKAI1) on lipopolysaccharides (LPS)-stimulatedRAW264.7 macrophage-like cells and mouse bone marrow-derived macrophages (BMDM) and to identify its underlyingmechanism. Our data showed that rhKAI1 suppressedexpression levels of classically macrophages (M1) phenotyperelatedsurface markers F4/80+CD86+ in LPS-stimulated BMDMand RAW264.7 cells. In addition, LPS markedly increased mRNAexpression and release levels of pro-inflammatory cytokinesand mediators such as interleukin (IL)-1β, IL-6, tumor necrosisfactor-α, cyclooxygenase-2, nitric oxide and prostaglandin E2,whereas these increases were substantially down-regulated byrhKAI1. Furthermore, LPS strongly increased expression of NF-κBp65 in the nuclei and phosphorylation of ERK, JNK, and p38MAPK. However, nuclear translocation of NF-κB p65 and phosphorylationof JNK were greatly reversed in the presence ofrhKAI1. Especially, rhKAI1 markedly suppressed expression oftoll-like receptor (TLR4) and prevented binding of LPS withTLR4 through molecular docking predict analysis. Importantly,Glu 214 of rhKAI1 residue strongly interacted with Lys 360 ofTLR4 residue, with a binding distance of 2.9 Å. Taken together,these findings suggest that rhKAI1 has an anti-inflammatoryeffect on LPS-polarized macrophages by interacting withTLR4 and down-regulating the JNK/NF-κB signaling pathway.
Abstract
KAI1/CD82, a membrane tetraspanin protein, can prevent variouscancers and retinal disorders through its anti-angiogenicand anti-metastatic capacity. However, little is known about itsanti-inflammatory effect and molecular mechanism. Therefore,the present study aimed to inLPSvestigate effect of a recombinantprotein of the large extracellular domain of human KAI1(Gly 111-Leu 228, rhKAI1) on lipopolysaccharides (LPS)-stimulatedRAW264.7 macrophage-like cells and mouse bone marrow-derived macrophages (BMDM) and to identify its underlyingmechanism. Our data showed that rhKAI1 suppressedexpression levels of classically macrophages (M1) phenotyperelatedsurface markers F4/80+CD86+ in LPS-stimulated BMDMand RAW264.7 cells. In addition, LPS markedly increased mRNAexpression and release levels of pro-inflammatory cytokinesand mediators such as interleukin (IL)-1β, IL-6, tumor necrosisfactor-α, cyclooxygenase-2, nitric oxide and prostaglandin E2,whereas these increases were substantially down-regulated byrhKAI1. Furthermore, LPS strongly increased expression of NF-κBp65 in the nuclei and phosphorylation of ERK, JNK, and p38MAPK. However, nuclear translocation of NF-κB p65 and phosphorylationof JNK were greatly reversed in the presence ofrhKAI1. Especially, rhKAI1 markedly suppressed expression oftoll-like receptor (TLR4) and prevented binding of LPS withTLR4 through molecular docking predict analysis. Importantly,Glu 214 of rhKAI1 residue strongly interacted with Lys 360 ofTLR4 residue, with a binding distance of 2.9 Å. Taken together,these findings suggest that rhKAI1 has an anti-inflammatoryeffect on LPS-polarized macrophages by interacting withTLR4 and down-regulating the JNK/NF-κB signaling pathway.
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