Protocatechualdehyde Inhibits the Osteoclast Differentiation of RAW264.7 and BMM Cells by Regulating NF-κB and MAPK Activity.

Protocatechualdehyde (PCA), an important component of Salvia miltiorrhiza, has many activities, such as anti-inflammatory and antisepsis activities. However, the role of PCA in osteoclasts is not clear. We used RAW264.7 cells (a mouse leukemic monocyte/macrophage cell line) and bone marrow macrophages (BMMs) to probe the role of PCA in osteoclasts and the underlying mechanism. The effects of PCA on cell activity were evaluated with CCK-8 assays. TRAP staining detected mature osteoclasts. Corning Osteo Assay Surface plates were used to examine absorption. The levels of RNA and protein were analyzed, respectively, using RT-PCR and Western blotting. PCA (5 µg/ml) was not toxic to the two cell types but reduced the formation of osteoclasts and bone absorption. Furthermore, PCA restrained the expression of mRNAs encoding proteins associated with osteoclasts and reduced the phosphorylation of proteins in important signaling pathways. The results indicate that PCA inhibits osteoclast differentiation by suppressing NF-κB and MAPK activity.

FKBP4 integrates FKBP4/Hsp90/IKK with FKBP4/Hsp70/RelA complex to promote lung adenocarcinoma progression via IKK/NF-κB signaling.

FKBP4 belongs to the family of immunophilins, which serve as a regulator for steroid receptor activity. Thus, FKBP4 has been recognized to play a critical role in several hormone-dependent cancers, including breast and prostate cancer. However, there is still no research to address the role of FKBP4 on lung adenocarcinoma (LUAD) progression. We found that FKBP4 expression was elevated in LUAD samples and predicted significantly shorter overall survival based on TCGA and our cohort of LUAD patients. Furthermore, FKBP4 robustly increased the proliferation, metastasis, and invasion of LUAD in vitro and vivo. Mechanistic studies revealed the interaction between FKBP4 and IKK kinase complex. We found that FKBP4 potentiated IKK kinase activity by interacting with Hsp90 and IKK subunits and promoting Hsp90/IKK association. Also, FKBP4 promotes the binding of IKKγ to IKKß, which supported the facilitation role in IKK complex assembly. We further identified that FKBP4 TPR domains are essential for FKBP4/IKK interaction since its association with Hsp90 is required. In addition, FKBP4 PPIase domains are involved in FKBP4/IKKγ interaction. Interestingly, the association between FKBP4 and Hsp70/RelA favors the transport of RelA toward the nucleus. Collectively, FKBP4 integrates FKBP4/Hsp90/IKK with FKBP4/Hsp70/RelA complex to potentiate the transcriptional activity and nuclear translocation of NF-κB, thereby promoting LUAD progression. Our findings suggest that FKBP4 may function as a prognostic biomarker of LUAD and provide a newly mechanistic insight into modulating IKK/NF-κB signaling.

[Expression of human HMGB1 A box in E. coli and its inhibitory effects on monocytes].

AIM: To clone human high mobility guoup box1 A box (HMGB1 A box) and express it in escherichia coli effectly, investigate the inhibit effection of the purpose protern to the activation of monocytes stimulated by immunocomplex. METHODS: According to human HMGB1 gene order which was optimized by our laboratory the PCR primer was designed which containing restriction enzyme cutting site. The HMGB1 A box gene was cloned following the whole gene synthesis template of human HMGB1, then the PCR product was inserted into clone vector pMD19-T. The positive colone was identified by colony PCR, zymography analysis and DNA sequencing. Recombinant colne vector was digested by restriction enzymes Nde I and Xho I and separated by agarose gel electrophoresis, then the fragment was inserted into the corresponding sites of expression vector pQE-T7-2. The positive recombinant expression vector was identified by colony PCR and the recombinant strains was induced by IPTG, then the purpose protein was identified by SDS-PAGE and Western blot. The recombinant protein of human HMGB1 A box was purificated by Ni(2+)-NTA chromatography and the inhibit effection of the purpose protern to the activation of monocyte stimulated by immunocomplex was identified by RT-PCR. RESULTS: We acquired expression strains of recombinant human HMGB1 A box, the target protein account for up to 40% of the whole protein of E.coli. Western blot showed recombinant protein can specificly reacted with anti-human HMGB1 polyclonal antibody and anti-His-Tag polyclonal antibody.The purpose protein was found more than 90% after purified, and can effectively inhibit the production of BAFF, IFN-gamma and TNF-alpha in monocyte which were induced by IC. CONCLUSION: A recombinant bacterial strain for expressing human HMGB1A box with biological activities was constructed successfully.