Salivary histatin 3 inhibits heat shock cognate protein 70-mediated inflammatory cytokine production through toll-like receptors in human gingival fibroblasts.
Salivary histatin 3 inhibits heat shock cognate protein 70-mediated inflammatory cytokine production through toll-like receptors in human gingival fibroblasts.
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DOI：
10.1186/1476-9255-11-4
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发表时间：
2014-02-04
期刊：
影响因子：
--
通讯作者：
Wang PL
中科院分区：
文献类型：
--
作者：
Imamura Y;Wang PL
唾液组胺素是一种与先天免疫系统相关的生物活性肽，具有抗菌活性。然而，人们对组蛋白针对宿主细胞的生理和生物学功能及其在口腔细胞炎症中的作用知之甚少。组胺素3与热休克同源蛋白70（HSC 70，组成型表达的热休克蛋白（HSP））结合。目前尚不清楚HSC 70是否参与口腔细胞的炎症反应。受损的口腔细胞释放一些细胞内蛋白质，包括HSC 70。释放的HSC 70可能诱导Toll样受体（TLR）激活，就像细胞外HSP 70（应激诱导HSP）一样，而组胺抑制素3影响这一过程。因此，我们测试了HSC 70激活TLR信号传导和组胺素3抑制这种激活和炎性细胞因子产生的假设。将核因子（NF）-κ B依赖性荧光素酶报告质粒转染到稳定表达TLR 2与辅助受体CD 14（293-TLR 2/CD 14细胞）或稳定表达TLR 4与CD 14和辅助分子MD 2（293-TLR 4/MD 2-CD 14细胞）的HEK 293细胞中。在存在或不存在组胺素3的情况下，用HSC 70刺激细胞，并使用荧光素酶测定进行检查。我们还刺激人牙龈成纤维细胞（HGFs）与HSC 70有或没有组胺素3。然后，我们分析了培养基中炎性细胞因子（白细胞介素（IL）-6和IL-8）的水平。用丝裂原活化蛋白激酶（MAPK）抗体和NF-κB抑制剂IκB-α抗体分别进行酶联免疫吸附试验（ELISA）和蛋白质印迹分析。使用有限的V8蛋白酶蛋白水解分析HSC 70的组胺素3结合形式。HSC 70在293-TLR 2/CD 14和293-TLR 4/MD 2-CD 14细胞中以剂量依赖性方式诱导NF-κB活化，histatin 3抑制该过程，当histatin 3与HSC 70结合被15-deoxyspergualin阻断时，其增强NF-κ B触发的活化。在HGF中，histatin 3还抑制HSC 70诱导的炎性细胞因子产生、细胞外信号调节蛋白激酶磷酸化和IκB-α降解。此外，与对照肽存在下的HSC 70相比，在组胺素3存在下的HSC 70对V8蛋白酶的消化具有相对抗性。组胺素3可能是HSC 70触发的TLR信号传导和炎性细胞因子产生的激活的抑制剂，并且可能参与口腔细胞中注意到的炎症过程。
Salivary histatins are bioactive peptides related to the innate immune system associated with antimicrobial activities. However, very little is known about the physiological and biological functions of histatins against host cells or their role in oral cell inflammation. Histatin 3 binds to heat shock cognate protein 70 (HSC70, a constitutively expressed heat shock protein (HSP)). It is unclear whether HSC70 is involved in the inflammatory response in oral cells. Injured oral cells release some intracellular proteins including HSC70. It is possible that released HSC70 induces toll-like receptor (TLR) activation, just as extracellular HSP70 (a stress inducible HSP) does, and that histatin 3 affects this process. Therefore, we tested the hypothesis that HSC70 activates TLR signaling and histatin 3 inhibits this activation and inflammatory cytokine production. A nuclear factor (NF)-κB-dependent luciferase reporter plasmid was transfected into HEK293 cells stably expressing TLR2 with coreceptor CD14 (293-TLR2/CD14 cells) or stably expressing TLR4 with CD14 and the accessory molecule MD2 (293-TLR4/MD2-CD14 cells). The cells were stimulated with HSC70 in the presence or absence of histatin 3, and examined using luciferase assays. We also stimulated human gingival fibroblasts (HGFs) with HSC70 with or without histatin 3. Then, we analyzed the levels of inflammatory cytokines (interleukin (IL)-6 and IL-8) in the culture media. Cell proteins were analyzed using enzyme-linked immunosorbent assay and Western blotting with antibodies of mitogen-activated protein kinases and NF-κB inhibitor IκB-α, respectively. Histatin 3-bound form of HSC70 was analyzed using limited V8 protease proteolysis. HSC70 induced NF-κB activation in a dose-dependent manner in 293-TLR2/CD14 and 293-TLR4/MD2-CD14 cells, and histatin 3 inhibited this process and when histatin 3 binding to HSC70 was precluded by 15-deoxyspergualin, which augmented NF-κB-triggered activation. In HGFs, histatin 3 also inhibited HSC70-induced inflammatory cytokine production, extracellular signal-regulated protein kinase phosphorylation, and degradation of IκB-α. Moreover, HSC70 in the presence of histatin 3 was relatively resistant to digestion by V8 protease compared with HSC70 in the presence of control peptide. Histatin 3 may be an inhibitor of HSC70-triggered activation of TLR signaling and inflammatory cytokine production and may be involved in inflammation processes noted in oral cells.
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