LRP16 Integrates into NF-κB Transcriptional Complex and Is Required for Its Functional Activation

Background: Nuclear factor kappa B (NF-kappa B)-mediated pathways have been widely implicated in cell survival, development and tumor progression. Although the molecular events of determining NF-kappa B translocation from cytoplasm to nucleus have been extensively documented, the regulatory mechanisms of NF-kappa B 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-kappa B coactivator UXT. Here, we investigated the regulatory role of LRP16 on NF-kappa B activation. Methodology: GST pull-down and coimmunoprecipitation (CoIP) assays assessed protein-protein interactions. The functional activity of NF-kappa B 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-kappa B were performed on primary human gastric carcinoma samples. Results: We demonstrate that LRP16 integrates into NF-kappa B transcriptional complex through associating with its p65 component. RNA interference knockdown of the endogenous LRP16 in cells leads to impaired NF-kappa B activity and significantly attenuated NF-kappa B-dependent gene expression. Mechanistic analysis revealed that knockdown of LRP16 did not affect tumor necrosis factor a (TNF-alpha)-induced nuclear translocation of NF-kappa B, but blunted the formation or stabilization of functional NF-kappa B/p300/CREB-binding protein transcription complex in the nucleus. In addition, knockdown of LRP16 also sensitizes cells to apoptosis induced by TNF-alpha. Finally, a positive link between LRP16 expression intensity in nuclei of tumor cells and NF-kappa B activity was preliminarily established in human gastric carcinoma specimens. Conclusions: Our findings not only indicate that LRP16 is a crucial regulator for NF-kappa B activation inside the nucleus, but also suggest that LRP16 may be an important contributor to the aberrant activation of NF-kappa B in tumors.