title: LRP16 integrates into NF-κB transcriptional complex and is required for its functional activation.
abstract: BACKGROUND
  Nuclear factor κB (NF-κB)-mediated pathways have been widely implicated in cell survival, development and tumor progression. Although the molecular events of determining NF-κB translocation from cytoplasm to nucleus have been extensively documented, the regulatory mechanisms of NF-κB activity inside the nucleus are still poorly understood. Being a special member of macro domain proteins, LRP16 was previously identified as a coactivator of both estrogen receptor and androgen receptor, and as an interactor of NF-κB coactivator UXT. Here, we investigated the regulatory role of LRP16 on NF-κB activation.
  
  
  METHODS
  GST pull-down and coimmunoprecipitation (CoIP) assays assessed protein-protein interactions. The functional activity of NF-κB was assessed by luciferase assays, changes in expression of its target genes, and its DNA binding ability. Annexin V staining and flow cytometry analysis were used to evaluate cell apoptosis. Immunohistochemical staining of LRP16 and enzyme-linked immunosorbent assay-based evaluation of active NF-κB were performed on primary human gastric carcinoma samples.
  
  
  RESULTS
  We demonstrate that LRP16 integrates into NF-κB transcriptional complex through associating with its p65 component. RNA interference knockdown of the endogenous LRP16 in cells leads to impaired NF-κB activity and significantly attenuated NF-κB-dependent gene expression. Mechanistic analysis revealed that knockdown of LRP16 did not affect tumor necrosis factor α (TNF-α)-induced nuclear translocation of NF-κB, but blunted the formation or stabilization of functional NF-κB/p300/CREB-binding protein transcription complex in the nucleus. In addition, knockdown of LRP16 also sensitizes cells to apoptosis induced by TNF-α. Finally, a positive link between LRP16 expression intensity in nuclei of tumor cells and NF-κB activity was preliminarily established in human gastric carcinoma specimens.
  
  
  CONCLUSIONS
  Our findings not only indicate that LRP16 is a crucial regulator for NF-κB activation inside the nucleus, but also suggest that LRP16 may be an important contributor to the aberrant activation of NF-κB in tumors.
authors.affiliation: Department of Molecular Biology, Institute of Basic Medicine, Chinese PLA General Hospital, Beijing, China.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Wu
authors.forename: Yazhuo
authors.initials: Y
authors.lastname: Li
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Li
authors.forename: Dongdong
authors.initials: D
authors.lastname: Ti
authors.forename: Yali
authors.initials: Y
authors.lastname: Zhao
authors.forename: Yiling
authors.initials: Y
authors.lastname: Si
authors.forename: Qian
authors.initials: Q
authors.lastname: Mei
authors.forename: Po
authors.initials: P
authors.lastname: Zhao
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Fu
authors.forename: Weidong
authors.initials: W
authors.lastname: Han
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D017209:Apoptosis / Q000187:drug effects; D002265:Carboxylic Ester Hydrolases; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D016328:NF-kappa B; D009363:Neoplasm Proteins; D014409:Tumor Necrosis Factor-alpha; D002265:Carboxylic Ester Hydrolases; C476832:MACROD1 protein, human